Immunotherapy against melanoma and other cancers

ABSTRACT

A method of treating a patient who has melanoma includes administering to said patient a composition containing a population of activated T cells that selectively recognize cells in the patient that aberrantly express a peptide. A pharmaceutical composition contains activated T cells that selectively recognize cells in a patient that aberrantly express a peptide, and a pharmaceutically acceptable carrier, in which the T cells bind to the peptide in a complex with an MHC class I molecule, and the composition is for treating the patient who has melanoma. A method of treating a patient who has melanoma includes administering to said patient a composition comprising a peptide in the form of a pharmaceutically acceptable salt, thereby inducing a T-cell response to the melanoma.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/848,523, filed 20 Dec. 2017, which is a continuation of U.S.application Ser. No. 15/638,786, filed 30 Jun. 2017, now U.S. Pat. No.9,901,629, which is a continuation of U.S. application Ser. No.15/489,399, filed 17 Apr. 2017, now U.S. Pat. No. 10,035,838, whichclaims the benefit of U.S. Provisional Application Ser. No. 62/325,773,filed 21 Apr. 2016, and Great Britain Application No. 1606919.7, filed21 Apr. 2016, the content of each of these applications is hereinincorporated by reference in their entirety.

This application also is related to PCT/EP2017/059016 filed 13 Apr.2017, the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-068005_ST25.txt” createdon 15 Oct. 2018, and 52,386 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION Melanoma

Globally, melanoma is diagnosed with an incidence rate of 3.0 in100,000, representing 1.7% of all cancer cases. In 2012, 232,000 womenwere diagnosed with melanoma. The mortality rate of 0.7 in 100,000 womenis substantially lower than the incidence rate (Ferlay et al., 2013).The lifetime risk of getting melanoma is about 2.4% (1 in 40) forwhites, 0.1% (1 in 1,000) for blacks, and 0.5% (1 in 200) for Hispanics.Although the average age at melanoma diagnosis is 62, it is one of themost common cancers in young adults (especially young women) (AmericanCancer Society, 2015).

For patients with localized melanoma, prognosis is good with adequatesurgical excision which is reflected by the relatively low melanomamortality rates (World Cancer Report, 2014). In line, the 5-yearsurvival rate is more than 90% and 80% for stage I and II lesions,respectively (Kaufman et al., 2013).

Metastatic melanoma is however largely resistant to current therapies(World Cancer Report, 2014). The 5-year survival rate is 78-40% forstage IIIA-C and 15-20% for stage IV (American Cancer Society, 2015).

Besides sun-exposure, the risk to develop melanoma is influenced byother environmental factors such as age and sex as well as anatomicallocation and individual susceptibility. Ultraviolet-emitting tanningdevices also increase the risk of malignant melanoma. In 20-40% offamilies with a melanoma history, CDKN2A mutations have been found(World Cancer Report, 2014).

Melanomas occur primarily in the skin—more than 95% of cases—but arealso found in the mucous membranes of the mouth, nose, anus, and vaginaand, to a lesser extent, the intestine. Furthermore, melanocytes arepresent in the conjunctiva, the retina, and the meninges. Melanoma canbe subtyped histologically into superficial spreading melanoma, nodularmelanoma, acral lentiginous melanoma, and lentigo maligna melanoma.Melanomas are classified according to the TNM classification. Asrecommended in the American Joint Committee on Cancer staging manual,melanoma patients are categorized into three groups: localized diseasewith no evidence of metastases (stage I-II), regional disease (stageIII), and distant metastatic disease (stage IV) (World Cancer Report,2014).

The standard therapy in melanoma is complete surgical resection withsurrounding healthy tissue. If resection is not complete or not possibleat all, patients receive primary radiation therapy, which can becombined with interferon-alpha administration in advanced stages (stagesIIB/C and IIIA-C). In Germany no standard therapeutic regimen exists forthe treatment of patients with late stage and metastasizing melanoma.Therefore, patients suffering from late stage and metastasizing melanomashould be treated in the context of a clinical study. Therapeuticoptions include mono-chemotherapy, poly-chemotherapy and targetedtherapies with specific inhibitors. Dacarbazine, temozolamide andfotemustin are currently used in mono-chemotherapy trial. Differentcombinations of chemotherapeutics are investigated in poly-chemotherapystudies: the CarboTax regimen (carboplatin plus paclitaxel), the GemTreoregimen (gemcitabine plus treosulfan), the DVP regimen (dacarbazine plusvindesin plus cisplatin), the BHD regimen (carmustine plus hyroxyureaplus dacarbazine) and the BOLD regimen (bleomycin plus vincristine pluslomustine plus darcarbazine). Furthermore, chemotherapy in combinationwith ipilimumab and the administration of specific BRAF, c-KIT and N-RASinhibitors to patients with mutations within the respective genes arecurrently evaluated in clinical trials (S3-Leitlinie Melanom, 2013).Enhancing the anti-tumor immune responses appears to be a promisingstrategy for the treatment of advanced melanoma. In the United Statesthe immune checkpoint inhibitor ipilimumab as well as the BRAF kinaseinhibitors vemurafenib and dabrafenib and the MEK inhibitor trametinibare already approved for the treatment of advanced melanoma. Bothapproaches increase the patient's anti-tumor immunity—ipilimumabdirectly by reducing T cell inhibition and the kinase inhibitorsindirectly by enhancing the expression of melanocyte differentiationantigens (Srivastava and McDermott, 2014). Vemurafenib has a responserate of 40-50% but with a median duration of only 5-6 months (WorldCancer Report, 2014). Furthermore, the combination of vemurafenib withof cobimetinib, another MAPK pathway inhibitor targeting the kinase MEKreceived FDA approval (National Cancer Institute, 2015).

Several different vaccination approaches have already been evaluated inpatients with advanced melanoma. So far, phase III trials revealedrather disappointing results and vaccination strategies clearly need tobe improved.

Adoptive T cell transfer shows great promise for the treatment ofadvanced stage melanoma. In vitro expanded autologous tumor infiltratinglymphocytes as well as T cells harboring a high affinity T cell receptorfor the cancer-testis antigen NY-ESO-1 had significant beneficial andlow toxic effects upon transfer into melanoma patients. Unfortunately, Tcells with high affinity T cell receptors for the melanocyte specificantigens MART1 and gp100 and the cancer-testis antigen MAGEA3 inducedconsiderable toxic effects in clinical trials. Thus, adoptive T celltransfer has high therapeutic potential, but safety and tolerability ofthese treatments needs to be further increased (Phan and Rosenberg,2013; Hinrichs and Restifo, 2013).

Only recently, the FDA approved the first oncolytic virus therapy,talimogene laherparepvec (T-VEC). The agency approved T-VEC for thetreatment of some patients with metastatic melanoma that cannot besurgically removed (National Cancer Institute, 2015).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and melanoma in particular. There is alsoa need to identify factors representing biomarkers for cancer in generaland melanoma in particular, leading to better diagnosis of cancer,assessment of prognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes. T-helper cells, activated by MHC class II epitopes,play an important role in orchestrating the effector function of CTLs inanti-tumor immunity. T-helper cell epitopes that trigger a T-helper cellresponse of the TH1 type support effector functions of CD8-positivekiller T cells, which include cytotoxic functions directed against tumorcells displaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-1-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 237 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 237, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 237 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 237,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table2 bind to HLA-A*02. The peptides in Table 2 were identified from largelistings as results of high-throughput screenings with high error ratesor calculated using algorithms, but have not been associated with cancerat all before. The peptides in Table 3 are additional peptides that maybe useful individually or in combination with the other peptides of theinvention. The peptides in Table 4 are furthermore useful in thediagnosis and/or treatment of various other malignancies that involve anover-expression or over-presentation of the respective underlyingpolypeptide.

TABLE 1 Peptides according to the present invention. SEQ ID No SequenceGene ID(s) Official Gene Symbol(s) 1 FLDVKELML 6271 S100A1 2 VLLGENVEL83872 HMCN1 3 VLFKDPVSV 2134 EXTL1 4 KTWDQVPFSV 6490 PMEL 5 ILDEGHILQL83872 HMCN1 6 SIPDTIASV 283652 SLC24A5 7 NLQEKVPEL 200728 TMEM17 8SIIPYLLEA 89797 NAV2 9 SLAGLVLYV 399694 SHC4 10 KMTQYITEL 9915 ARNT2 11TLIELLLPKL 6773 STAT2 12 RLDDKTTNV 5027 P2RX7 13 IQSETTVTV 83872 HMCN114 VLYEMLYGL 100533105, 23678, C8orf44-SGK3, SGK3, SGK1 6446 15VLYDPVVGC 11180 WDR6 16 GLFPSNFVTA 8027 STAM 17 GVVHGVATV 6622 SNCA 18SLADVVDTL 55553, 6660 SOX6, SOX5 19 VLAVLGAVVAV 3106, 3107 HLA-B, HLA-C20 VISPHGIASV 5270 SERPINE2 21 FMYNFQLVTL 2181 ACSL3 22 KLLELQELVL 2801,342096, 55149, GOLGA6D, GOLGA2, 55889, 653641, 653643 MTPAP, GOLGA6A,GOLGA6C, GOLGA6B 23 FLGDPPPGL 127703 C1orf216 24 SLVAILHLL 55742 PARVA25 FIDPEQIQV 101060422, 8515 LOC101060422, ITGA10 26 KIEDLIKYL 11258DCTN3 27 TLWYVPLSL 11332 ACOT7 28 IVDNTTMQL 3421 IDH3G 29 ILDDVAMVL58517 RBM25 30 VLFPMDLAL 5784 PTPN14 31 FLPRKFPSL 23246, 727967 BOP1 32GLDIITNKV 54802 TRIT1 33 SLYSYFQKV 51151 SLC45A2 34 YLINFEIRSL 57539WDR35 35 ALFAAGANV 116211, 255758 TM4SF19 36 SVNGFISTL 3709, 3710 ITPR2,ITPR3 37 TLKEYLESL 285190, 400966, 5903, RANBP2, RGPD1, RGPD2, 653489,727851, RGPD3, RGPD4, RGPD5, 729540, 729857, 84220 RGPD6, RGPD8 38KLGFGTGVNVYL 55872 PBK 39 ALPPPPASI 342184 FMN1 40 LLSNTVSTL 283652SLC24A5 41 LLDDPTNAHFI 2118 ETV4 42 VLKADVVLL 259307 IL4I1 43 LLPDPLYSL9631 NUP155 44 FLYTYIAKV 54763 ROPN1 45 FVYGEPREL 392555, 51438 MAGEC246 VMSSTLYTV 51151 SLC45A2 47 ALDSDPVGL 25894 PLEKHG4 48 HLIGWTAFL 51151SLC45A2 49 ALLSQDFEL 4241 MFI2 50 HLDQIFQNL 6355 CCL8 51 LIDKIIEYL 25914RTTN 52 NLDYAILKL 374393 FAM111B 53 ILDEEKFNV 55127 HEATR1 54 LLDSGAFHL27304 MOCS3 55 NLDKLYHGL 8318 CDC45 56 ILDELVKSL 56852 RAD18 57GILSFLPVL 2213, 9103 FCGR2B, FCGR2C 58 ILGDWSIQV 135228 CD109 59IIDDVMKEL 79959 CEP76 60 ILPEAQDYFL 80071 CCDC15 61 KLSVHVTAL 89858SIGLEC12 62 LLDTTQKYL 54811 ZNF562 63 SIDDSDPIV 26046 LTN1 64 SLGPIMLTKI2086 ERV3-1 65 TTLGGFAKV 196528 ARID2 66 VMFEYGMRL 23279 NUP160 67YVDSEGIVRM 11169 WDHD1 68 FLAEAARSL 79654 HECTD3 69 IIDDKPIGL 9420CYP7B1 70 LIDEAAQML 85441 HELZ2 71 SLDEVAVSL 144455 E2F7 72 TLLEVDAIVNA140733 MACROD2 73 ELDKIYETL 51163 DBR1 74 GTIPLIESL 160418 TMTC3 75FMYAGQLTL 79842 ZBTB3 76 QIDSIHLLL 55102 ATG2B 77 SIDDVVKKL 6672 SP10078 ALKDLVNLI 23001 WDFY3 79 AVDNILLKL 1763 DNA2 80 FADELSHLL 79830 ZMYM181 FLDDGNQML 79659 DYNC2H1 82 GIDDLHISL 23224 SYNE2 83 GLDKVITVL 9833MELK 84 GLDTILQNL 79830 ZMYM1 85 GLLDVMYQV 254251 LCORL 86 HTLPHEIVVNL23195 MDN1 87 IIDPPLHGQLL 80144 FRAS1 88 ILDGIIREL 254065 BRWD3 89ILDNSPAFL 163786 SASS6 90 ILDYIHNGL 84640 USP38 91 ILLDRLFSV 54796, 646BNC1, BNC2 92 KLPGFPTQDDEV 51202 DDX47 93 LLAKAVQNV 100271927, 10156RASA4, RASA4B 94 LLDAFSIKL 23224 SYNE2 95 LLDALQHEL 93323 HAUS8 96LLDMSLVKL 55038 CDCA4 97 NLDATVTAL 22995 CEP152 98 NLPNTNSILGV 57862ZNF410 99 NLPSELPQL 100137047, 100137049, JMJD7 8681 100 NLREILQNV253260 RICTOR 101 NVDENVAEL 51678 MPP6 102 RLPDQFSKL 51735, 96459RAPGEF6 103 SLDAVMPHL 6477 SIAH1 104 SLDQIIQHL 51750, 8771 RTEL1 105SLKQTVVTL 8924 HERC2 106 TLSEICEFI 2297, 2306 FOXD1, FOXD2 107 TLVAFLQQV79659 DYNC2H1 108 TVIRPLPGL 389524, 84163 GTF2IRD2, GTF2IRD2B 109VIDDLIQKL 79659 DYNC2H1 110 VLDTLTKVL 26292 MYCBP 111 VLDVSFNRL 2811GP1BA 112 VLPAVLTRL 2175 FANCA 113 VLYSLVSKI 23335 WDR7 114 VVDDIVSKL10926 DBF4 115 YIDDVFMGL 84002 B3GNT5 116 LMDETMKEL 348 APOE 117KQQASQVLV 5627 PROS1 118 TMIEICEKL 10988 METAP2 119 SLGLGFISRV 4644MYO5A 120 QLMEGKVVL 27340 UTP20 121 FLEDLVPYL 84342 COG8 122 YVDDFGVSV2132 EXT2 123 LLGEGIPSA 85461 TANC1 124 FLPQKIIYL 5721 PSME2 125YLFAFLNHL 23380, 57522, 9901 SRGAP1, SRGAP3, SRGAP2 126 SLIDFVVTC 10457GPNMB 127 TLISDIEAVKA 81619 TSPAN14 128 ALFPGDVDRL 5834 PYGB 129VLPDDLSGV 2771 GNAI2 130 GLVDVLYTA 9710 KIAA0355 131 FVDPNGKISL 8729GBF1 132 FLDASGAKL 9689 BZW1 133 ALDPAYTTL 3172 HNF4A 134 LLDEVLHTM 4089SMAD4 135 FLDDQETRL 10906 TRAFD1 136 FAYDGKDYIAL 3105, 3106, 3107 HLA-C,HLA-B, HLA-A 137 ILPSNLLTV 5297 PI4KA 138 YLDKTFYNL 23325 KIAA1033 139AVDATVNQV 10130 PDIA6 140 RLEAYLARV 10763 NES 141 YVIDPIKGL 5339 PLEC142 FVDGSAIQV 26010 SPATS2L 143 ILDDSALYL 23130 ATG2A 144 SVDEVEISV10598 AHSA1 145 TLPNIYVTL 55102 ATG2B 146 GVGPVPARA 81533 ITFG1 147ILDDQTNKL 1601 DAB2 148 TLKDIVQTV 54855 FAM46C 149 YLDTFALKL 401548SNX30 150 KLFPSPLQTL 111 ADCY5 151 FLGEPASYLYL 6638 SNRPN 152 IMEDFTTFL55601 DDX60 153 RLDEVSREL 6238 RRBP1 154 TLGTATFTV 5321 PLA2G4A 155GLAGFFASV 2030 SLC29A1 156 ALMDTDGSGKLNL 825 CAPN3 157 HLFETISQA 5691PSMB3 158 KLIPSIIVL 719 C3AR1 159 TILATVPLV 6720 SREBF1 160 ALDDISESI25996 REXO2 161 GLCDSIITI 23788 MTCH2 162 TLDGNPFLV 929 CD14 163RLMANPEALKI 2633 GBP1 164 ALFFQLVDV 6185 RPN2 165 ALIEVLQPLI 7453 WARS166 SIIPPLFTV 6748 SSR4 167 KVLGDVIEV 1410 CRYAB 168 KLLAATLLL 10673TNFSF13B 169 TLLESIQHV 8924 HERC2 170 KLKEAVEAI 8450 CUL4B 171 KVSGVILSV1186 CLCN7 172 FLPAGIVAV 11319 ECD 173 ALDDIIYRA 84668 FAM126A 174TLLEGLTEL 8382 NME5 175 VLDSVDVRL 113189 CHST14 176 TLYEQEIEV 23127GLT25D2 177 ILWDTLLRL 29954 POMT2 178 FAYDGKDYIA 3105, 3106, 3107 HLA-A,HLA-B, HLA-C 179 ALDDTVLQV 337876 CHSY3 180 KLAEALYIA 22938 SNW1 181GLIDLEANYL 222553 SLC35F1 182 SVALVIHNV 10385 BTN2A2 183 FLDSLIYGA 55974SLC50A1 184 VLFSSPPVILL 5621 PRNP 185 ILADATAKM 7094 TLN1 186 FLDHEMVFL100996782, 54797 LOC100996782, MED18 187 SLPRPTPQA 1601 DAB2

TABLE 2 Additional peptides according to the present invention SEQ ID NoSequence Gene ID(s) Official Gene Symbol(s) 188 VVVDPIQSV 10213 PSMD14189 KALQFLEEV 908 CCT6A 190 RLVSLITLL 57231 SNX14 191 YLDKMNNNI 9686VGLL4 192 KLFTQIFGV 27434 POLM 193 ALDEPTTNL 10111 RAD50 194 TLDDIMAAV26057 ANKRD17 195 IAAGIFNDL 5695 PSMB7 196 ALEPIDITV 5885 RAD21 197ALDSGFNSV 84859 LRCH3 198 EVVDKINQV 23224 SYNE2 199 AIHTAILTL 5683 PSMA2200 LLEEINHFL 472 ATM 201 SLIDRTIKM 84928 TMEM209 202 RVAFKINSV 91543RSAD2 203 FLNEDISKL 22989 MYH15 204 RMDEEFTKI 728689, 8663 EIF3C, EIF3CL205 SLKSKVLSV 122830 NAA30 206 LLYEDIPDKV 22920 KIFAP3 207 VQIGDIVTV6205 RPS11 208 YSDDIPHAL 3646 EIF3E 209 SILDGLIHL 55705 IPO9 210LLPELRDWGV 56931 DUS3L 211 FLPFLTTEV 55974 SLC50A1 212 LLKDSIVQL 5573PRKAR1A 213 LLDPTNVFI 119559 SFXN4 214 VLMEMSYRL 55159 RFWD3 215EVISKLYAV 10694 CCT8 216 TLLHFLAEL 1729 DIAPH1 217 NMMSGISSV 1457CSNK2A1 218 STLHLVLRL 6233, 728590, 7311, UBC, RPS27A, UBA52, UBB, 7314,7316 RPS27AP11 219 FLDSEVSEL 64151 NCAPG 220 SAAEPTPAV 29803 REPIN1 221SLLPTEQPRL 65057 ACD 222 LLSEIEEHL 1653 DDX1 223 FLETNVPLL 1495, 1496CTNNA2, CTNNA1 224 ILDEPTNHL 55324 ABCF3 225 VLFGAVITGA 100507703, 3105LOC100507703, HLA-A 226 VLNEYFHNV 1175 AP2S1 227 FLLEQEKTQAL 11277,84126 TREX1, ATRIP 228 FLNLFNHTL 28962 OSTM1 229 LLEPFVHQV 51447 IP6K2230 HLDEARTLL 56254 RNF20 231 KMVGDVTGA 10410, 10581, 8519 IFITM2,IFITM1, IFITM3 232 KILPDLNTV 9875 URB1 233 QLYNQIIKL 6731 SRP72 234KVPEIEVTV 2969, 2970 GTF2I, GTF2IP1 235 ALADLQEAV 85461 TANC1 236GLDSGFHSV 4034 LRCH4 237 VLYNESLQL 56254 RNF20

TABLE 3 Peptides useful for, e.g., personalized cancer therapies SEQ IDNo Sequence Gene ID(s) Official Gene Symbol(s) 238 KLLDKPEQFL 342184FMN1 239 FLNDIFERI 337873, 337874 HIST2H2BC, HIST2H2BD 240 GLAEFQENV57405 SPC25 241 RLYTKLLNEA 4651 MYO10 242 SLESKLTSV 9289 GPR56 243ALAGIVTNV 11077 HSF2BP 244 ILLEKSVSV 80728 ARHGAP39 245 LLVDDSFLHTV253982 ASPHD1 246 TQDDYVLEV 5793, 5803 PTPRZ1, PTPRG 247 ALLNAILHSA25926 NOL11 248 GLFAGLGGAGA 10916 MAGED2 249 KLQDGLLHI 7076 TIMP1 250RVLPPSALQSV 9212 AURKB 251 VLDGKVAVV 6660 SOX5 252 YLLDMPLWYL 7153 TOP2A253 KLDIKVETV 55243 KIRREL 254 FLMKNSDLYGA 79801 SHCBP1 255 LLLGERVAL23475 QPRT 256 VLLDTILQL 11077 HSF2BP 257 VLLNEILEQV 64151 NCAPG 258FLKNELDNV 10293 TRAIP 259 GLDGIPFTV 7205 TRIP6 260 QLIDYERQL 11072DUSP14 261 GLSEVLVQI 57553 MICAL3 262 KLAVALLAA 3576 IL8 263 YALDLSTFL8870 IER3 264 KVFDEVIEV 8908 GYG2 265 ILYDLQQNL 3783 KCNN4 266 YLAPENGYL6625 SNRNP70 267 LLTDNVVKL 79810 PTCD2 268 ALADLSVAV 3363 HTR7 269ALNESLVEC 55165 CEP55 270 KIWEELSVLEV 4102, 4105 MAGEA3, MAGEA6 271SLVQRVETI 1894 ECT2 272 YLDPLWHQL 2072 ERCC4 273 ALSELLQQV 9816 URB2 274RLHDENILL 23322 RPGRIP1L 275 SLLNQPKAV 63967 CLSPN 276 FLDSQITTV 255119C4orf22 277 KTASINQNV 81930 KIF18A 278 SLITGQDLLSV 51804 SIX4 279VVAAHLAGA 148113 CILP2 280 LLWPSSVPA 246777, 79400 SPESP1, NOX5 281GLLENSPHL 25788 RAD54B 282 LLIPFTIFM 1237 CCR8 283 YTFSGDVQL 4312 MMP1284 TIGIPFPNV 83990 BRIP1 285 YLMDDFSSL 1293 COL6A3 286 GLNGFNVLL 144455E2F7 287 KISDFGLATV 1111 CHEK1 288 ALLEQTGDMSL 1063 CENPF 289 ILAQDVAQL24137 KIF4A 290 NVAEIVIHI 83540 NUF2 291 LLDDIFIRL 143570 XRRA1 292ALGDKFLLRV 4608 MYBPH 293 FLDGRPLTL 83734 ATG10 294 FLLAEDTKV 10592 SMC2295 FLPQPVPLSV 57695 USP37 296 FTAEFLEKV 79801 SHCBP1 297 GVDDAFYTL 3845KRAS 298 KLQEEIPVL 1062 CENPE 299 NLLIDDKGTIKL 983 CDK1 300 QIDDVTIKI64151 NCAPG 301 RVIDDSLVVGV 2187 FANCB 302 TVLQELINV 3832 KIF11 303KLGDFGLLVEL 9088 PKMYT1 304 VLLAQIIQV 89797 NAV2 305 TLLKTIIKV 57545CC2D2A 306 KMLDEILLQL 5425 POLD2 307 ALAGGITMV 790 CAD 308 KLLSDPNYGV79188 TMEM43 309 MQKEITAL 440915, 58, 59, 60, POTEKP, ACTA1, ACTA2,644936, 70, 71, 72 ACTB, ACTC1, ACTG1, ACTG2 310 ALASVIKEL 28981 IFT81311 KLMDYIDEL 85444 LRRCC1 312 TAVGHALVL 1293 COL6A3 313 LLLDTVTMQV22820 COPG1 314 SLFEWFHPL 2519 FUCA2 315 KLSWDLIYL 51148 CERCAM 316ALAELLHGA 26470 SEZ6L2 317 NLAEELEGV 10763 NES 318 SIIEYLPTL 79915 ATAD5319 ALSSSQAEV 3833 KIFC1 320 KIIGIMEEV 2956 MSH6 321 YLPTFFLTV 54898ELOVL2 322 SLHFLILYV 487, 488 ATP2A1, ATP2A2 323 VVDKTLLLV 53838C11orf24 324 SLANNVTSV 131566 DCBLD2 325 VLVDDDGIKVV 79022 TMEM106C 326ALSGTLSGV 4174 MCM5 327 ALADKELLPSV 84883 AIFM2 328 SLSQELVGV 24149ZNF318 329 VLAPRVLRA 5954 RCN1 330 KMFFLIDKV 4599 MX1 331 ALSQVTLLL392636 AGMO 332 AVVEFLTSV 29102 DROSHA 333 RIPAYFVTV 7407 VARS 334VLLDKIKNLQV 1293 COL6A3 335 KLASMLETL 112464 PRKCDBP 336 YVDPVITSI 4233MET 337 FLVDGSSAL 1293 COL6A3 338 SLNKWIFTV 339665 SLC35E4

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, acute myelogenousleukemia, breast cancer, bile duct cancer, brain cancer, chroniclymphocytic leukemia, colorectal carcinoma, esophageal cancer,gallbladder cancer, gastric cancer, hepatocellular cancer, non-Hodgkinlymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer,prostate cancer, renal cell cancer, small cell lung cancer, urinarybladder cancer and uterine cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 237. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 34 (see Table 1), and their uses in theimmunotherapy of melanoma, acute myelogenous leukemia, breast cancer,bile duct cancer, brain cancer, chronic lymphocytic leukemia, colorectalcarcinoma, esophageal cancer, gallbladder cancer, gastric cancer,hepatocellular cancer, non-Hodgkin lymphoma, non-small cell lung cancer,ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer,small cell lung cancer, urinary bladder cancer and uterine cancer, andpreferably melanoma.

As shown in the following Tables 4A and 4B, many of the peptidesaccording to the present invention are also found on other tumor typesand can, thus, also be used in the immunotherapy of other indications.Also refer to FIGS. 1A-1J and Example 1.

TABLE 4A Peptides according to the present invention and their specificuses in other proliferative diseases, especially in other cancerousdiseases. The table shows for selected peptides on which additionaltumor types they were found and either over-presented on more than 5% ofthe measured tumor samples, or presented on more than 5% of the measuredtumor samples with a ratio of geometric means tumor vs normal tissuesbeing larger than 3. Over-presentation is here defined as higherpresentation on the tumor sample as compared to the normal sample withhighest presentation. Normal tissues against which over-presentation wastested were: adipose tissue, adrenal gland, blood cells, blood vessel,bone marrow, brain, esophagus, eye, gallbladder, heart, kidney, largeintestine, liver, lung, lymph node, nerve, pancreas, parathyroid gland,peritoneum, pituitary, pleura, salivary gland, skeletal muscle, skin,small intestine, spleen, stomach, thymus, thyroid gland, trachea,ureter, urinary bladder. SEQ ID No. Sequence relevant organs/diseases 1FLDVKELML RCC, HCC, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer2 VLLGENVEL NHL, BRCA 7 NLQEKVPEL PC, AML, BRCA, Uterine Cancer 8SIIPYLLEA Uterine Cancer 10 KMTQYITEL Brain Cancer 11 TLIELLLPKL CLL 15VLYDPVVGC CLL, NHL, AML, Uterine Cancer 17 GVVHGVATV AML, Urinarybladder cancer 18 SLADVVDTL Brain Cancer, CLL, NHL, Uterine Cancer 19VLAVLGAVVAV SCLC, RCC, BRCA, Uterine Cancer 20 VISPHGIASV Brain Cancer,Uterine Cancer 21 FMYNFQLVTL SCLC, Urinary bladder cancer 22 KLLELQELVLNSCLC, Brain Cancer, CRC, BRCA, OC 23 FLGDPPPGL CLL, NHL, AML, BRCA,Urinary bladder cancer, Uterine Cancer 24 SLVAILHLL NHL, GallbladderCancer, Bile Duct Cancer 27 TLWYVPLSL CLL, NHL, AML, Uterine Cancer 29ILDDVAMVL CLL, NHL 30 VLFPMDLAL RCC 31 FLPRKFPSL NSCLC, CRC, CLL, NHL,Esophageal Cancer, OC, Urinary bladder cancer, Uterine Cancer 32GLDIITNKV NHL 36 SVNGFISTL AML 57 GILSFLPVL CLL, NHL 80 FADELSHLL AML116 LMDETMKEL NSCLC, Brain Cancer, HCC, NHL, BRCA, OC, Urinary bladdercancer, Gallbladder Cancer, Bile Duct Cancer 118 TMIEICEKL NSCLC, AML,OC 119 SLGLGFISRV BRCA 120 QLMEGKVVL NHL 121 FLEDLVPYL CLL, NHL, AML 122YVDDFGVSV AML 123 LLGEGIPSA Urinary bladder cancer, Uterine Cancer 124FLPQKIIYL GC, BRCA, OC, Uterine Cancer 125 YLFAFLNHL AML, OC, UterineCancer 126 SLIDFVVTC RCC, PC, NHL, OC, Uterine Cancer 127 TLISDIEAVKACLL, NHL, Urinary bladder cancer, Uterine Cancer 128 ALFPGDVDRL BrainCancer, GC, CRC, PC, PrC, BRCA, Esophageal Cancer, Urinary bladdercancer 130 GLVDVLYTA NSCLC, RCC, Brain Cancer, BRCA, Esophageal Cancer,Uterine Cancer 133 ALDPAYTTL HCC, CLL, NHL, AML, Uterine Cancer 135FLDDQETRL SCLC, CLL, OC 138 YLDKTFYNL CRC, CLL, AML 139 AVDATVNQV CLL,Uterine Cancer 143 ILDDSALYL NHL, Uterine Cancer 144 SVDEVEISV CLL 145TLPNIYVTL NHL, AML 146 GVGPVPARA PC, AML, Urinary bladder cancer 148TLKDIVQTV CLL, NHL, BRCA 150 KLFPSPLQTL SCLC, RCC, PrC, GallbladderCancer, Bile Duct Cancer 151 FLGEPASYLYL NHL 154 TLGTATFTV Urinarybladder cancer, Uterine Cancer 155 GLAGFFASV HCC, NHL, BRCA, EsophagealCancer, Urinary bladder cancer, Uterine Cancer 157 HLFETISQA Urinarybladder cancer 158 KLIPSIIVL AML 159 TILATVPLV SCLC, NHL, AML, BRCA,Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile DuctCancer 160 ALDDISESI Esophageal Cancer 161 GLCDSIITI NSCLC, BrainCancer, PC, NHL, BRCA, Uterine Cancer 163 RLMANPEALKI NHL, OC, Urinarybladder cancer, Uterine Cancer 164 ALFFQLVDV SCLC, RCC, AML, BRCA 165ALIEVLQPLI Urinary bladder cancer 166 SIIPPLFTV SCLC, PC, AML, BRCA, OC,Urinary bladder cancer 167 KVLGDVIEV RCC, Brain Cancer 168 KLLAATLLLRCC, AML, Esophageal Cancer 169 TLLESIQHV SCLC, Brain Cancer, CRC, NHL,AML, BRCA, OC 170 KLKEAVEAI RCC, CLL, NHL 171 KVSGVILSV NHL, AML, BRCA172 FLPAGIVAV CLL, NHL, AML, Urinary bladder cancer, Uterine Cancer 173ALDDIIYRA CLL, NHL 174 TLLEGLTEL OC, Uterine Cancer 175 VLDSVDVRL RCC,AML 176 TLYEQEIEV RCC, Brain Cancer, PC, PrC, NHL 177 ILWDTLLRL RCC,AML, Gallbladder Cancer, Bile Duct Cancer 178 FAYDGKDYIA BRCA,Esophageal Cancer, OC 179 ALDDTVLQV SCLC, Esophageal Cancer 180KLAEALYIA PrC, BRCA, Esophageal Cancer, Urinary bladder cancer 181GLIDLEANYL Brain Cancer, CLL, Uterine Cancer 182 SVALVIHNV NHL 183FLDSLIYGA AML, BRCA, Uterine Cancer 184 VLFSSPPVILL NSCLC, Brain Cancer,PrC, CLL, NHL, Esophageal Cancer, Urinary bladder cancer 186 FLDHEMVFLCLL, NHL, AML, Urinary bladder cancer 187 SLPRPTPQA RCC 189 KALQFLEEVGC, CRC, BRCA, Uterine Cancer 190 RLVSLITLL CLL 191 YLDKMNNNI NSCLC,RCC, Brain Cancer, PC, NHL, AML, BRCA, Esophageal Cancer, Urinarybladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 192KLFTQIFGV HCC 193 ALDEPTTNL AML, Urinary bladder cancer, GallbladderCancer, Bile Duct Cancer 194 TLDDIMAAV NSCLC, SCLC, RCC, Brain Cancer,CRC, CLL, NHL, AML, BRCA, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer 195 IAAGIFNDL CLL, AML 196ALEPIDITV BRCA 197 ALDSGFNSV CLL, NHL, Uterine Cancer 198 EVVDKINQV RCC199 AIHTAILTL CRC, BRCA 203 FLNEDISKL RCC 206 LLYEDIPDKV CLL, NHL,Esophageal Cancer, OC, Urinary bladder cancer 207 VQIGDIVTV GC, AML,BRCA 208 YSDDIPHAL AML 209 SILDGLIHL CLL, NHL, AML 210 LLPELRDWGV NHL211 FLPFLTTEV HCC, CLL, NHL, AML, OC, Uterine Cancer 212 LLKDSIVQL RCC,CLL, Urinary bladder cancer 213 LLDPTNVFI PrC, NHL, AML, Urinary bladdercancer 214 VLMEMSYRL SCLC, RCC, CRC, CLL, NHL, AML, BRCA, Urinarybladder cancer, Gallbladder Cancer, Bile Duct Cancer 215 EVISKLYAV BRCA,Urinary bladder cancer 216 TLLHFLAEL CLL, NHL 217 NMMSGISSV BrainCancer, CRC, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer,Bile Duct Cancer 218 STLHLVLRL RCC, GC, HCC, PC 219 FLDSEVSEL NHL, AML,Urinary bladder cancer, Uterine Cancer 220 SAAEPTPAV Gallbladder Cancer,Bile Duct Cancer 221 SLLPTEQPRL NSCLC, SCLC, Brain Cancer, CRC, HCC,PrC, CLL, NHL, Esophageal Cancer, Urinary bladder cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer 222 LLSEIEEHL CLL 223FLETNVPLL Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 224ILDEPTNHL CLL 225 VLFGAVITGA SCLC, Brain Cancer, HCC, PC, CLL, NHL, AML,BRCA, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer 226VLNEYFHNV SCLC, HCC, BRCA, Esophageal Cancer, Urinary bladder cancer,Uterine Cancer 227 FLLEQEKTQAL PrC, CLL, NHL, BRCA, Esophageal Cancer,OC 228 FLNLFNHTL CLL 229 LLEPFVHQV CLL, NHL, Urinary bladder cancer,Uterine Cancer 230 HLDEARTLL CLL, NHL, AML, Uterine Cancer 232 KILPDLNTVBrain Cancer, Urinary bladder cancer 233 QLYNQIIKL CLL, NHL 234KVPEIEVTV NHL, AML, Uterine Cancer 235 ALADLQEAV Brain Cancer, PrC,Uterine Cancer 236 GLDSGFHSV PC, NHL, BRCA 237 VLYNESLQL NHL NSCLC= non-small cell lung cancer, SCLC = small cell lung cancer, RCC= kidney cancer, CRC = colon or rectum cancer, GC = stomach cancer, HCC= liver cancer, PC = pancreatic cancer, PrC = prostate cancer, leukemia,BRCA = breast cancer, OC = ovarian cancer, NHL = non-Hodgkin lymphoma,AML = acute myelogenous leukemia CLL = chronic lymphatic leukemia

TABLE 4B Peptides according to the present invention and their specificuses in other proliferative diseases, especially in other cancerousdiseases. The table shows for selected peptides on which additionaltumor types they were found and either over-presented on more than 5% ofthe measured tumor samples, or presented on more than 5% of the measuredtumor samples with a ratio of geometric means tumor vs normal tissuesbeing larger than 3. Over-presentation is defined as higher presentationon the tumor sample as compared to the normal sample with highestpresentation. Normal tissues against which over-presentation was testedwere: adipose tissue, adrenal gland, artery, bone marrow, brain, centralnerve, colon,, esophagus, eye, gallbladder, heart, kidney, liver, lung,lymph node, white blood cells, pancreas, parathyroid gland, peripheralnerve, peritoneum, pituitary, pleura, rectum, salivary gland, skeletalmuscle, skin, small intestine, spleen, stomach, thymus, thyroid gland,trachea, ureter, urinary bladder, vein. SEQ ID No Sequence AdditionalEntities 1 FLDVKELML Brain Cancer, OC 7 NLQEKVPEL HNSCC 8 SIIPYLLEA HCC,CLL, NHL, HNSCC 13 IQSETTVTV HNSCC 14 VLYEMLYGL HNSCC 15 VLYDPVVGC HNSCC16 GLFPSNFVTA CLL, BRCA, AML 17 GVVHGVATV NHL 19 VLAVLGAVVAV HCC, CLL,HNSCC 20 VISPHGIASV Esophageal Cancer, HNSCC 22 KLLELQELVL HNSCC 23FLGDPPPGL Gallbladder Cancer, Bile Duct Cancer, HNSCC 24 SLVAILHLLNSCLC, OC, HNSCC 27 TLWYVPLSL HNSCC 28 IVDNTTMQL GC, AML 30 VLFPMDLALHNSCC 31 FLPRKFPSL HNSCC 37 TLKEYLESL HCC, Esophageal Cancer 50HLDQIFQNL GC 52 NLDYAILKL GC 54 LLDSGAFHL GC 56 ILDELVKSL GC 58ILGDWSIQV HNSCC 59 IIDDVMKEL AML 62 LLDTTQKYL AML 64 SLGPIMLTKI HCC 68FLAEAARSL HCC 73 ELDKIYETL GC 76 QIDSIHLLL GC 78 ALKDLVNLI HCC 79AVDNILLKL GC 82 GIDDLHISL GC 84 GLDTILQNL GC, AML 88 ILDGIIREL GC 91ILLDRLFSV HNSCC 94 LLDAFSIKL GC 100 NLREILQNV HCC 102 RLPDQFSKL AML 104SLDQIIQHL AML 105 SLKQTVVTL HCC 109 VIDDLIQKL AML 114 VVDDIVSKL GC, AML115 YIDDVFMGL GC 120 QLMEGKVVL HNSCC 122 YVDDFGVSV GC 123 LLGEGIPSAHNSCC 124 FLPQKIIYL NHL 126 SLIDFVVTC NSCLC, HCC, HNSCC 129 VLPDDLSGVHCC 131 FVDPNGKISL Urinary bladder cancer, AML 132 FLDASGAKL GC 134LLDEVLHTM GC 135 FLDDQETRL HNSCC 136 FAYDGKDYIAL RCC, GallbladderCancer, Bile Duct Cancer, NHL 137 ILPSNLLTV HCC, CLL, Urinary bladdercancer, Uterine Cancer, AML, NHL 141 YVIDPIKGL Esophageal Cancer 142FVDGSAIQV GC 143 ILDDSALYL AML 146 GVGPVPARA HCC, NHL 148 TLKDIVQTV HCC150 KLFPSPLQTL Urinary bladder cancer 151 FLGEPASYLYL HCC, BRCA, OC,Uterine Cancer 153 RLDEVSREL GC 159 TILATVPLV GC, HNSCC 160 ALDDISESIGC, Uterine Cancer 161 GLCDSIITI GC, CRC, HCC, HNSCC 163 RLMANPEALKIHCC, HNSCC 164 ALFFQLVDV HCC, HNSCC 165 ALIEVLQPLI GC, HNSCC 166SIIPPLFTV HCC 168 KLLAATLLL HCC 169 TLLESIQHV HNSCC 170 KLKEAVEAI HCC173 ALDDIIYRA HNSCC 175 VLDSVDVRL GC 178 FAYDGKDYIA RCC, HNSCC 179ALDDTVLQV HNSCC 180 KLAEALYIA HNSCC 182 SVALVIHNV RCC, GC, HCC 184VLFSSPPVILL HNSCC 186 FLDHEMVFL Uterine Cancer 189 KALQFLEEV HCC 192KLFTQIFGV SCLC 193 ALDEPTTNL CRC, CLL, NHL 195 IAAGIFNDL GallbladderCancer, Bile Duct Cancer 198 EVVDKINQV GC 203 FLNEDISKL HCC 204RMDEEFTKI AML 205 SLKSKVLSV HCC 206 LLYEDIPDKV RCC, HCC, HNSCC 207VQIGDIVTV HNSCC 208 YSDDIPHAL Gallbladder Cancer, Bile Duct Cancer 210LLPELRDWGV HCC, CLL 211 FLPFLTTEV SCLC, HNSCC 212 LLKDSIVQL GC, HCC,AML, NHL 213 LLDPTNVFI GC, OC, Esophageal Cancer, HNSCC 214 VLMEMSYRLNSCLC, HNSCC 215 EVISKLYAV RCC, GC, HCC 217 NMMSGISSV BRCA, HNSCC 219FLDSEVSEL SCLC, GC 220 SAAEPTPAV HCC 221 SLLPTEQPRL HNSCC 224 ILDEPTNHLUterine Cancer 225 VLFGAVITGA HNSCC 226 VLNEYFHNV NHL, HNSCC 227FLLEQEKTQAL HNSCC 230 HLDEARTLL GC 232 KILPDLNTV HCC 234 KVPEIEVTV GC,CLL, BRCA, OC, Gallbladder Cancer, Bile Duct Cancer 236 GLDSGFHSV CLL,AML 237 VLYNESLQL CLL, AML

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 19, 30, 126, 130, 136, 150, 164, 167, 168, 170,175, 176, 177, 178, 182, 187, 191, 194, 198, 203, 206, 212, 214, 215,and 218 for the—in one preferred embodiment combined—treatment of RCC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 8, 19, 37, 64, 68, 78, 100, 105, 116, 126, 129,133, 135, 137, 146, 148, 151, 155, 161, 163, 164, 166, 168, 170, 182,189, 192, 203, 205, 206, 210, 211, 212, 215, 218, 220, 221, 225, 226,and 232 for the—in one preferred embodiment combined—treatment of HCC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 7, 8, 15, 18, 19, 20, 23, 27, 31, 123, 124, 125,126, 127, 130, 133, 137, 139, 143, 151, 154, 155, 159, 160, 161, 163,172, 174, 181, 183, 186, 189, 191, 194, 197, 211, 217, 219, 221, 223,224, 225, 226, 229, 230, 234, and 235 for the—in one preferredembodiment combined—treatment of uterine cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 23, 24, 116, 136, 150, 159, 177, 191, 193, 194,195, 208, 214, 217, 220, 221, 223, and 234 for the—in one preferredembodiment combined—treatment of gallbladder cancer, and/or bile ductcancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 2, 8, 11, 15, 17, 18, 23, 24, 27, 29, 31, 32, 57, 116,120, 121, 124, 126, 127, 133, 135, 136, 137, 138, 139, 143, 144, 145,146, 148, 151, 155, 159, 161, 163, 169, 170, 171, 172, 173, 176, 181,182, 184, 186, 190, 191, 193, 194, 195, 197, 206, 209, 210, 211, 212,213, 214, 216, 219, 221, 222, 224, 225, 226, 227, 228, 229, 230, 233,234, 236, and 237 for the—in one preferred embodiment combined—treatmentof NHL.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 2, 7, 16, 19, 22, 23, 116, 119, 124, 128, 130, 148,151, 155, 159, 161, 164, 166, 171, 178, 180, 183, 189, 191, 194, 196,199, 207, 214, 215, 217, 225, 226, 227, 234 and 236 for the—in onepreferred embodiment combined—treatment of BRCA.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 7, 126, 128, 146, 161, 166, 176, 191, 218, 225, and236 for the—in one preferred embodiment combined—treatment of PC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 7, 15, 16, 17, 23, 27, 28, 36, 59, 62, 80, 84, 102,104, 109, 114, 118, 121, 122, 125, 131, 133, 137, 138, 143, 145, 146,158, 159, 164, 166, 168, 169, 171, 172, 175, 177, 183, 186, 191, 193,194, 195, 204, 207, 208, 209, 211, 212, 213, 214, 219, 225, 230, 234,236, and 237 for the—in one preferred embodiment combined—treatment ofAML.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 10, 18, 20, 22, 116, 128, 130, 161, 167, 169, 176,181, 184, 191, 194, 217, 221, 225, 232, and 235 for the—in one preferredembodiment combined—treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 17, 21, 23, 31, 116, 123, 127, 128, 131, 137, 146,150, 154, 155, 157, 159, 163, 165, 166, 172, 180, 184, 186, 191, 193,194, 206, 212, 213, 214, 215, 217, 219, 221, 225, 226, 229, and 232 forthe—in one preferred embodiment combined—treatment of urinary bladdercancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 19, 21, 135, 150, 159, 164, 166, 169, 179, 192, 194,211, 214, 219, 221, 225, and 226 for the—in one preferred embodimentcombined—treatment of SCLC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 22, 24, 31, 116, 118, 126, 130, 161, 184, 191, 194,214, and 221 for the—in one preferred embodiment combined—treatment ofNSCLC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 22, 24, 31, 116, 118, 124, 125, 126, 135, 151, 163,166, 169, 174, 178, 206, 211, 213, 227, and 234 for the—in one preferredembodiment combined—treatment of OC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 20, 31, 37, 128, 130, 141, 155, 160, 168, 178, 179,180, 184, 191, 206, 213, 221, 225, 226, and 227 for the—in one preferredembodiment combined—treatment of esophageal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 28, 50, 52, 54, 56, 73, 76, 79, 82, 84, 88, 94, 114,115, 122, 124, 128, 132, 134, 142, 153, 159, 160, 161, 165, 175, 182,189, 198, 207, 212, 213, 215, 218, 219, 230, and 234 for the—in onepreferred embodiment combined—treatment of GC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 22, 31, 128, 138, 161, 169, 189, 193, 194, 199, 214,217, and 221 for the—in one preferred embodiment combined—treatment ofCRC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 128, 150, 176, 180, 184, 213, 221, 227, and 235 forthe—in one preferred embodiment combined—treatment of PrC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 7, 8, 13, 14, 15, 19, 20, 22, 23, 24, 27, 30, 31, 58,91, 120, 123, 126, 135, 159, 161, 163, 164, 165, 169, 173, 178, 179,180, 184, 206, 207, 211, 213, 214, 217, 221, 225, 226, and 227 forthe—in one preferred embodiment combined—treatment of HNSCC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 8, 16, 19, 137, 193, 210, 234, 236, and 237 for the—inone preferred embodiment combined—treatment of CLL.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofmelanoma, acute myelogenous leukemia, breast cancer, bile duct cancer,brain cancer, chronic lymphocytic leukemia, colorectal carcinoma,esophageal cancer, gallbladder cancer, gastric cancer, hepatocellularcancer, non-Hodgkin lymphoma, non-small cell lung cancer, ovariancancer, pancreatic cancer, prostate cancer, renal cell cancer, smallcell lung cancer, urinary bladder cancer and uterine cancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 237.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No. 237, preferably containing SEQ IDNo. 1 to SEQ ID No. 34, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are melanoma, acute myelogenousleukemia, breast cancer, bile duct cancer, brain cancer, chroniclymphocytic leukemia, colorectal carcinoma, esophageal cancer,gallbladder cancer, gastric cancer, hepatocellular cancer, non-Hodgkinlymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer,prostate cancer, renal cell cancer, small cell lung cancer, urinarybladder cancer and uterine cancer, and preferably melanoma cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably melanoma. Themarker can be over-presentation of the peptide(s) themselves, orover-expression of the corresponding gene(s). The markers may also beused to predict the probability of success of a treatment, preferably animmunotherapy, and most preferred an immunotherapy targeting the sametarget that is identified by the biomarker. For example, an antibody orsoluble TCR can be used to stain sections of the tumor to detect thepresence of a peptide of interest in complex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

Both therapeutic and diagnostic uses against additional cancerousdiseases are disclosed in the following more detailed description of theunderlying expression products (polypeptides) of the peptides accordingto the invention.

ACOT7 has been found to be up-regulated in melanoma, where it may beinvolved in preventing lipotoxicity (Sumantran et al., 2015).

ACSL3 encodes for acyl-CoA synthetase long-chain family member 3. ACSL3is over-expressed in lung cancer and based on preclinical investigationis a promising new therapeutic target in lung cancer (Pei et al., 2013).The up-regulated expression of ACSL3 can serve as a potential biomarkerof estrogen receptor-specific breast cancer risk (Wang et al., 2013b).

APOE is involved in cholesterol transport and may be important inenabling tumor cell to fulfill their high cholesterol requirements. Itwas found to be over-expressed in various types of cancer such asgastric cancer, anaplastic thyroid carcinoma, prostate cancer andcolorectal cancer (Yasui et al., 2005; Ito et al., 2006; Sakashita etal., 2008; Shi et al., 2015b; Kang et al., 2016; Yencilek et al., 2016).Elevated serum levels of APOE were shown to be associated withmetastasis and poor prognosis in non-small cell lung cancer. Furthermorethey have been suggested as a prognostic marker in breast cancer and asa marker to monitor the efficiency of chemotherapy in small cell lungcancer (Shi et al., 2016; Xu et al., 2016b; Luo et al., 2016).

Loss of ARID2 through inactivating mutations was related to tumorprogression and recurrence in gastric cancer, hepatocellular carcinomaand non-small cell lung carcinoma (Manceau et al., 2013; You et al.,2015; Aso et al., 2015).

ARNT2 has been found to be over-expressed in non-small cell lung cancer,hepatocellular carcinoma, breast cancer and oral squamous cellcarcinoma. It acts as a tumor suppressor during cancer progressionbecause over-expression has been determined to increase overall survivaland promote cell apoptosis (Qin et al., 2011a; Li et al., 2015d; Yang etal., 2015; Kimura et al., 2016).

ATG2B encodes autophagy related 2B, a protein essential forautophagosome formation and regulation of lipid droplet volume anddistribution (Velikkakath et al., 2012). ATG2B frameshift mutations arecommon in gastric and colon carcinomas with high microsatelliteinstability (Kang et al., 2009).

ATM is a tumor suppressor which is frequently mutated in a broad rangeof human cancers including lung, colorectal, breast and hematopoieticcancers (Weber and Ryan, 2014). Loss of ATM has been associated with theincreased risk of various cancers including, breast, colorectal,prostate, lung and pancreatic ductal adenocarcinoma (Swift et al., 1987;Geoffroy-Perez et al., 2001; Angele et al., 2004; Roberts et al., 2012;Grant et al., 2013; Russell et al., 2015). Studies have shown that IL-8was able to rescue cell migration and invasion defects in ATM-depletedcells (Chen et al., 2015b). Low level of ATM protein was correlated withpoor metastasis-free survival in breast cancer patients. In addition,miR-203 and miR-421 over-expression may be involved in ATM de-regulationin these patients (Bueno et al., 2014; Rondeau et al., 2015).

BNC1 was shown to be part of a ten-gene methylation signature which washyper-methylated in colorectal adenomas and carcinomas (Patai et al.,2015). BNC1 was shown to be associated with prostate cancer since it wasfrequently methylated and thus inactivated in prostate cancer cell lines(Devaney et al., 2013). BNC1 was shown to be one of many potentialtargets that were aberrantly methylated in chronic lymphocytic leukemia,renal cell carcinoma and T-cell and B-cell childhood acute lymphoblasticleukemia (Tong et al., 2010; Morris et al., 2010; Dunwell et al., 2009).BNC1 was shown to play a role in the progression of primary breasttumors to brain metastases. Knock-down of BNC1 resulted in an increaseof migratory and invasive potential of breast cancer cell lines. Thus,BNC1 may be useful as a prognostic marker and a novel therapeutic target(Pangeni et al., 2015). BNC1 was shown to be associated with TGF-β1signaling (Feuerborn et al., 2015). BNC1 was shown to be associated withpoorer survival in clear cell renal cell carcinoma and poorer prognosisin renal cell carcinoma (Morris et al., 2010; Ricketts et al., 2014).BNC1 was shown to be frequently methylated in stage I invasivepancreatic cancers. Thus, BNC1 serves as a potential biomarker to detectearly-stage pancreatic cancer (Yi et al., 2013). BNC1 was shown to beup-regulated in squamous cell carcinomas of the head and neck (Boldrupet al., 2012). BNC1 was shown to be transcriptionally regulated by thep53-family member p63 in squamous cell carcinomas of the head and neck(Boldrup et al., 2012).

Several studies hit that BNC2 functions as a tumor suppressor gene inesophageal adenocarcinoma, ovarian cancer and glioblastoma. The gene isfrequently deleted and/or expression is reduced (Nord et al., 2009;Akagi et al., 2009; Cesaratto et al., 2016). BNC2 was found to bedown-regulated in hepatocellular carcinoma and also it was frequentlydeleted, which might be one important reason for its lower expressionlevel (Wu et al., 2016).

BOP1 is associated with ovarian cancer and colorectal cancer(Wrzeszczynski et al., 2011; Killian et al., 2006). BOP1 was shown to bea target gene of Wnt/β-catenin which induced EMT, cell migration andexperimental metastasis of colorectal cancer cells in mice. Thus, BOP1may serve as a therapeutic target in the treatment of colorectal cancermetastasis (Qi et al., 2015). BOP1 is associated with hepatocellularcarcinoma invasiveness and metastasis (Chung et al., 2011). BOP1 wasdescribed as a member of a molecular pathway associated with cell cyclearrest in a gastric cancer cell line upon treatment with mycophenolicacid, indicating a potential association of BOP1 with the anticanceractivity of the drug (Dun et al., 2013a; Dun et al., 2013b). BOP1 may bea possible marker for rectal cancer (Lips et al., 2008). BOP1 wasdescribed as a potential oncogene in ovarian cancer (Wrzeszczynski etal., 2011). BOP1 was shown to be up-regulated in hepatocellularcarcinoma (Chung et al., 2011). BOP1 was shown to be associated withmicrovascular invasion, shorter disease-free survival and metastasis inhepatocellular carcinoma (Chung et al., 2011). BOP1 was described as asubunit of the PeBoW complex, which is essential for cell proliferationand maturation of the large ribosomal subunit. Over-expression of BOP1was shown to inhibit cell proliferation (Rohrmoser et al., 2007).Expression of an amino-terminally truncated form of BOP1 resulted indown-regulation of G(1)-specific Cdk2 and Cdk4 kinase complexes,retinoblastoma and cyclin A while Cdk inhibitors p21 and p27 wereup-regulated. This led to an arrest in the G(1) phase (Pestov et al.,2001).

CAPN3 expression was found to be down-regulated in melanoma cells whichplay a role in the acquisition of a highly invasive phenotype (Huynh etal., 2009; Ruffini et al., 2013; Moretti et al., 2015). CAPN3 has beenshown to complex with Digestive-organ-expansion-factor (Dev) andtogether mediate degradation of tumor suppressor p53 (Zhu et al.,2014b).

CCT6A is associated with testicular germ cell tumors and malignantmelanomas (Tanic et al., 2006; Alagaratnam et al., 2011).

CCT8 was shown to be up-regulated in hepatocellular carcinoma (Huang etal., 2014c). CCT8 is associated with histologic grades, tumor size andpoor prognosis of hepatocellular carcinoma (Huang et al., 2014c).

RPI-1 and dasatinib treatment target CD109 to inhibit cancer cellproliferation (Caccia et al., 2011). CD109 is over-expressed innasopharyngeal carcinoma, laryngeal squamous cell carcinoma, non-smallcell lung cancer, pancreatic cancer, myxofibrosarcoma, esophagealsquamous cell carcinoma, head and neck cancer, and (triple-negative)breast cancer (Ni et al., 2012; Tao et al., 2014; Zhang et al., 2014a;Dong et al., 2015a; Emori et al., 2015; Haun et al., 2014; Hoover etal., 2015; Jia et al., 2016). CD109 might be used as prognosticbiomarker in nasopharyngeal carcinoma, vulvar squamous cell carcinoma,triple-negative breast cancer, hepatocellular carcinoma, and gallbladdersquamous cell/adenosquamous carcinoma. Secreted CD109 may be used asserum prognostic marker (Ye et al., 2016; Ozbay et al., 2013; Sakakuraet al., 2014; Tao et al., 2014; Dong et al., 2015b; Jia et al., 2016).CD109 is expressed on a rare group of circulating endothelial cellswhich may be used as prognostic marker in glioblastoma (Mancuso et al.,2014; Cuppini et al., 2013). Reduced expression of CD109 promotes tumorgrowth. It was shown to be down-regulated in uterine carcinosarcoma (Yeet al., 2016; Semczuk et al., 2013). CD109 promotes hepatocellularcarcinoma proliferation and is correlated with poor prognosis (Zong etal., 2016). CD109 over-expression is associated with surgical state,poor prognosis, and metastasis (Emori et al., 2013; Emori et al., 2015;Karhemo et al., 2012). CD109 inhibits TGF-beta1 signaling and promotesEGF signaling human glioblastoma cells (Man et al., 2012; Zhang et al.,2015).

CSNK2A1 has been shown to be involved in tumorigenesis byphosphorylating other proteins in breast cancer, colorectal cancer andgastric carcinoma. CSNK2A1 expression was shown to be an independentprognostic indicator for gastric carcinoma, breast cancer, and clearcell renal cell carcinoma (Kim et al., 2012; Bae et al., 2015; Kren etal., 2015; Rabjerg et al., 2016; Bae et al., 2016). CSNK2A1 has beensuggested as a therapeutic target in chronic myeloid leukemia andglioblastoma. Inhibiting Casein Kinase II as part of a proposed novelBCR-ABL/CK2/PTEN pathway promotes PTEN reactivation, which promotesapoptosis induction in cancer cells (Lee et al., 2013; Zheng et al.,2013; Morotti et al., 2015). CSNK2A1 was shown to be frequently mutatedin adult T-cell leukemia (Kataoka et al., 2015).

DYNC2H1 was shown to be up-regulated in glioblastoma multiforme (Yokotaet al., 2006).

EIF3E might play a role in the carcinogenesis of oral squamous cellcarcinoma (Yong et al., 2014). EIF3E is essential for proliferation andsurvival of glioblastoma cells (Sesen et al., 2014). EIF3E has anoncogenic role in breast cancer progression. Decreased EIF3E expressioncauses epithelial to mesenchymal transition in breast epithelial cells(Gillis and Lewis, 2013; Grzmil et al., 2010). EIF3E expression level issignificantly increased in bladder cancer (Chen et al., 2011). EIF3E isinvolved in non-small lung carcinoma (Marchetti et al., 2001).

Expression of human endogenous retroviruses (HERV) env proteins such asERV3-1 was shown to be significantly increased in the blood of primarybreast cancer patients, suggesting the potential use of HERV env genesas a diagnosis marker for primary breast cancer (Rhyu et al., 2014).ERV3-1 was shown to be significantly over-expressed in lessdifferentiated endometrial carcinoma, liver and lung tumor tissues(Strissel et al., 2012; Ahn and Kim, 2009). Loss of ERV3-1 mRNAexpression was described as being associated with susceptibility tochoriocarcinoma (Kato et al., 1988).

Epigenetic inactivation of EXTL1 has been found in leukemia andnon-melanoma cancer cells. In contrast, high expression of EXTL1 wasreported to be associated with poor prognosis in patients with multiplemyeloma. EXTL1 was shown to have altered N-glycosylation in humanaggressive breast cancer cell lines (Drake et al., 2012; Busse-Wicher etal., 2014). Deletion of EXTL1 was detected in several neuroblastoma andit was suggested as a tumor suppressor gene, but no clear evidence asfound of EXTL1 being involved in the causal investigation ofneuroblastoma (Mathysen et al., 2004).

FCGR2B is the pre-dominant Fc-receptor on B-cells and therefore a targetfor immunotherapy. Via activation of FCGR2B the monoclonal antibodyRituximab inhibits Kv1.3 channels that play an important role inmodulating lymphocyte proliferation and apoptosis, and induces apoptosisin human B lymphoma cells (Shah et al., 2013; Rankin et al., 2006; Wanget al., 2012). FCGR2B polymorphisms have been found to correlate withclinical response to specific immunotherapy such as rituximab andidiotype vaccination in follicular lymphoma. Also, polymorphisms inFCGR2B have been associated with binding affinity of natural killercells to trastuzumab, an antibody used in treatment of HER-positivebreast cancer (Musolino et al., 2008; Weng et al., 2009; Norton et al.,2014). FCGR2B expression prevents the lysis of human metastatic melanomacells by NK cell-mediated antibody-dependent cellular cytotoxicity,making it a marker of human metastatic melanoma (Cassard et al., 2008).

A correlation of FCGR2C polymorphisms and/or expression levels to theresponse to certain immunotherapies has been found in breast cancer,neck squamous cell carcinoma and metastatic renal cell carcinoma(Petricevic et al., 2013; Trivedi et al., 2016; Erbe et al., 2016).

A single nucleotide polymorphism in FMN1 is associated with an increasedrisk of prostate cancer (Lisitskaia et al., 2010).

The FLCN/FNIP1/FNIP2 complex regulates kidney cell proliferation rateand is functionally lost in the Birt-Hogg-Dubé syndrome which is ahereditary hamartoma syndrome (Schmidt and Linehan, 2015b; Schmidt andLinehan, 2015a; Hasumi et al., 2016). FNIP1 is involved in invariantnatural killer T cell development (Park et al., 2014). FNIP1 promoteslysosome recruitment and the Rag interactions of the tumor suppressorFLCN (Petit et al., 2013). FNIP1 is involved in mTORC1 activation viaRagC/D (Linehan et al., 2010; Tsun et al., 2013). FNIP1 is involved inkidney tumor suppression and may be used as therapeutic target (Hasumiet al., 2015).

FOXD1 has been shown to be over-expressed in breast cancer, clear cellsarcoma of the kidney, gastric cancer and Hodgkin lymphoma. Theover-expression may increase cell proliferation and has been suggestedas a therapeutic target. In gastric cancer and hepatocellular carcinomait has been found to be part of the transcriptional regulatory network,whose downstream target genes are involved in cancerogenesis (Nagel etal., 2014; Karlsson et al., 2014; Zhao et al., 2015b; Xu et al., 2016a;Chen et al., 2016). Up-regulated FOXD1 expression levels have beendetermined as a prognostic marker for poor outcome in non-small celllung cancer (Nakayama et al., 2015).

FOXD2 was found to be highly expressed in prostate cancer and lymph nodemetastases (Heul-Nieuwenhuijsen et al., 2009). FOXD2 has been shown tobe differently methylated in serrated adenocarcinoma compared to othercolorectal cancer types, suggesting it as a biomarker to identify thisparticular type of colorectal cancer (Conesa-Zamora et al., 2015).

GBF1 has been identified as a host factor that enhances adenoviruscancer cell killing. Cancer cells are susceptible to oncolytic viruses,making them a cancer treatment option, and GBF1 knock-down or chemicalinhibition enhances melanoma or epithelial cancer cell killing byadenovirus infection by triggering unfolded protein response (Prasad etal., 2014).

GNAI2 over-expression has been observed in ovarian cancer andhepatocellular carcinoma. More specifically, GNAI2 expression decreasedin early stage ovarian cancer, while it increased in advanced cancers,implicating GNAI2 as a critical regulator of oncogenesis and an upstreamdriver of cancer progression in ovarian cancer (Peters et al., 2005;Raymond, Jr. et al., 2014). GNAI2 expression is regulated bymicroRNA-138, that is frequently de-regulated in various cancers liketongue squamous cell carcinoma and in turn GNAI2 is up-regulated. GNAI2is also a functional target of miR-30d in hepatocellular carcinoma cells(Jiang et al., 2011; Yao et al., 2010).

In gastric cancer high GOLGA2 expression levels were found to have apositive correlation with the pathological differentiation and tumornode metastasis stage, and also predict shorter overall survival.Furthermore, GOLGA2 contributes to epithelial-mesenchymal transition byup-regulating the expression of SNAI1 (Zhao et al., 2015a). GOLGA2expression is progressively lost in colorectal cancer and the lossdisrupts the cells apical-basal polarity as well as front-rear polarityand may play affect other processes relevant for tumorigenesis(Baschieri and Farhan, 2015; Baschieri et al., 2015). GOLGA2 has beensuggested as a therapeutic target, because down-regulation decreasedangiogenesis and cell cancer invasion in tumorigenesis in lung cancer(Chang et al., 2012).

GOLGA6A is located on one of the regions which were found to inheritpolymorphisms in Patients with Paget's disease of bone (Chung and Van,2012). GOLGA6A was identified as a fusion partner for PAX5 being anearly player in leukemogenesis (Coyaud et al., 2010).

The HERC2/OCA2 region on chromosome 15q13.1 is one of several loci thatpredispose to cutaneous melanoma (Amos et al., 2011; Xiao et al., 2014).HERC2 regulates the stability of different DNA repair factors includingCHK1, p53 and BRCA1 (Bekker-Jensen et al., 2010; Cubillos-Rojas et al.,2014; Zhu et al., 2014a; Peng et al., 2015).

HLA-B reduced expression has been associated with poorer survival inesophageal cancer. However, in gastric and colorectal cancer, theprognostic value of HLA-B remains conflicting and it can be both up- anddown-regulated (Powell et al., 2012; Gallou et al., 2016).

HLA class I molecules are ligands for killer immunoglobulin likereceptors (KIR), that negatively regulate NK cells and T cells and lackof KIR-HLA interactions have been associated with potent NK-mediatedantitumor efficacy and increased survival in acute myeloid leukemia. Inovarian cancer and non-small cell lung cancer certain genotypes of HLA-Chave an effect cancer development (Romagne et al., 2009; Wisniewski etal., 2012; Giebel et al., 2014). Reduced expression of HLA-C has beenassociated with poorer survival in esophageal cancer. However, ingastric and colorectal cancer the prognostic value of HLA-C remainsconflicting and it can be both up- and down-regulated. In colorectalcancer most tumor cells mimic the HLA phenotypes of their normalcounterparts to evade NK-mediated immunosurveillance (Gao et al., 2013;Powell et al., 2012; Doubrovina et al., 2003; Benevolo et al., 2007).

HMCN1 was found to be up-regulated in human soft tissue tumors and mightrepresent a novel candidate biomarker and therapeutic target (Sarver etal., 2015). HMCN1 was found to be involved in skin development andepithelial morphogenesis and showed a down-regulated expression inmultiple drug-resistant ovarian cancer cells (Januchowski et al., 2014;Westcot et al., 2015). Furthermore, HMCN1 is related to cell polarityand somatically mutated in gastric and colorectal cancers (Lee et al.,2015).

IDH3G was found to be differentially expressed in gastric cancer andmight be associated with drug resistance (Zhou et al., 2015).

IL411 protein expression is very frequent in tumors. IL411 was detectedin tumor-associated macrophages of different tumor entities, inneoplastic cells from lymphomas and in rare cases of solid cancersmainly mesothelioma (Carbonnelle-Puscian et al., 2009). IL4I1up-regulation in human Th17 cells limits their T-cell receptor(TCR)-mediated expansion by blocking the molecular pathway involved inthe activation of the IL-2 promoter and by maintaining high levels ofTob1, which impairs entry into the cell cycle (Santarlasci et al.,2014).

IPO9 encodes the protein importin 9, which acts as a scaffolding proteinand is important in regulating cellular function in both the immunesystem and the nervous system, by activating signaling pathways like theRas/Erk pathway or by enhancing mitochondria-mediated apoptosis (Murrinand Talbot, 2007; Wang et al., 2002).

De-regulation of ITGA10 has been shown to be a down-stream effect of thede-regulation of other cancer genes like ERG in leukemia, miR-375 inlung cancer or EPHB4 in prostate cancer (Mertens-Walker et al., 2015;Mochmann et al., 2014; Jin et al., 2015). ITGA10 has been found to beunder-expressed in solid osteoblasts that have frequent inactivation ofthe pRb pathway (Engel et al., 2013).

Single nucleotide polymorphism in the ITPR2 gene were correlated withrisk of renal cell carcinoma in a Chinese population. Likewise, twocommon variants in linkage disequilibrium, rs718314 and rs1049380 in theITPR2 gene were identified as novel susceptibility loci for renal cellcarcinoma. Moreover, over-expression of ITPR2 was observed in normalacute myeloid leukemia patients compared to healthy persons (Wu et al.,2012; Shi et al., 2015a; Zhang et al., 2016b). In normal acute myeloidleukemia, elevated levels of ITPR2 expression was associated withshorter overall survival and event-free survival (Shi et al., 2015a).

ITPR3 is over-expressed in several cancer types including colorectal,gastric and breast cancer and directly related to cancer progression andthe aggressiveness of the tumor (Shibao et al., 2010; Mound et al.,2013; Sakakura et al., 2003). Akt can protect cells in anITPR3-dependent manner from apoptosis through reducing the Ca2+ releasefrom the endoplasmatic reticulum (Marchi et al., 2012).

Researchers have observed that the levels of mRNA expression for theKIFAP3 gene were significantly reduced in tumorous tissue samplesrelative to non-cancerous renal cortex tissue samples. Others reportedover-expression of KIFAP3 protein in breast cancers. Another group hasshown that the expression of the KIFAP3 gene was significantly changedbetween breast cancer cells treated with recombinant bromelain and thecontrol cells (Gotoh et al., 2014; Fouz et al., 2014; Telikicherla etal., 2012).

MACROD2 showed somatic alterations (often intragenic deletions) in livercancer, colorectal cancer, gastric cancer and esophageal squamous cellcarcinoma (Briffa et al., 2015; Tada et al., 2010; van den Broek et al.,2015; Hu et al., 2016; Fujimoto et al., 2016). MACROD2 increases p300binding to estrogen response elements in a subset of estrogenreceptor-alpha (ER) regulated genes and shows an increased expression inprimary breast tumors where it is associated with worse overall survival(Mohseni et al., 2014). The MACROD2 gene was found to be deleted invarious cancer types, but a tumor suppressor role of MACROD2 could notbe established (Rajaram et al., 2013). MACROD2 plays a role inMARylation and is able to ‘read’ and ‘erase’ this modification on targetproteins (Feijs et al., 2013).

Over-expression of MAGEC2 increases the level of cyclin E and promotesG1-S transition and cell proliferation (Hao et al., 2015). MAGEC2promotes proliferation and resistance to apoptosis in Multiple Myelomasuggesting that MAGEC2-specific immunotherapies have the potential toeradicate the most malignant cells (Lajmi et al., 2015). MAGEC2, anepithelial-mesenchymal transition inducer, is associated with breastcancer metastasis. Multivariate analyses showed that MAGEC2 expressionwas an independent risk factor for patient overall survival andmetastasis-free survival (Yang et al., 2014).

The above mentioned increased expression of METAP2 and the anti-cancereffect of METAPA2 inhibitors has been studied in various cancers,including non-small cell lung cancer, pilocytic astrocytoma, colon andcolorectal cancer and neuroblastoma (Morowitz et al., 2005; Selvakumaret al., 2009; Ho et al., 2013; Shimizu et al., 2016).

Although the exact biological functions of MFI2 remain elusive, agrowing number of roles have been attributed to the protein, includingiron transport/metabolism, angiogenesis, proliferation, cellularmigration and tumorigenesis. MFI2 over-expressing tumors have beensuggested as targets that are sensitive to antibody-drug conjugates(Dunn et al., 2006; Smith et al., 2006; Suryo et al., 2012). MIF2 levelshave been shown to be significantly increased at the plasma level ofcolorectal cancer, making it a potential serological marker. It may alsobe involved in transformation from benign tumor to malignancy and is amarker of an invasive tumor phenotype (Shin et al., 2014;Dus-Szachniewicz et al., 2015).

It has been reported that MTCH2 is a suppressed by miR-135b, that isup-regulated in breast cancers and it seems that miR-135b and itstargets, MIDI and MTCH2, are relevant coordinators of mammary glandtumor progression (Arigoni et al., 2013). MTCH2 seems to be involved inrapid ABT-737 induced apoptosis in lymphoma and primary leukemia cells.ABT-737 induces MTCH2, resulting in mitochondrial matrix swelling andrupture of the outer mitochondrial membrane (Vogler et al., 2008).

Antibodies to poly(A) polymerase were observed in serum samples fromhuman patients with leukemia, polycythemia vera and Wilms tumor (Stetleret al., 1981).

MYO5A was shown to be associated with a novel trafficking pathway inmelanoma that promotes tumor resistance through Akt2/MYO5A activation(Fernandez-Perez et al., 2013). MYO5A was up-regulated in invasivenon-functioning pituitary adenomas and may thus serve as a useful markerof tumor invasiveness (Galland et al., 2010). MYO5A mRNA expression wasincreased in a number of highly metastatic cancer cell lines andmetastatic colorectal cancer tissues. Furthermore, suppression of MYO5Ain those cancer cells impede their migration and metastasis capabilitiesboth in vitro and in vivo (Lan et al., 2010). MYO5A was shown to beapplicable in a four-gene model for the identification occult nodalmetastasis in oral squamous cell carcinoma (Mendez et al., 2011).

NAA30 plays an important role in growth and survival ofglioblastoma-initiating cells possibly by regulating hypoxia response(HIF1α), levels of p-MTOR (Ser2448) and the p53 pathway (Mughal et al.,2015). NAA30 is differentially expressed during development or incarcinomas of higher eukaryotes and is thus suggested to be more highlyexpressed in cells undergoing rapid protein synthesis (Polevoda andSherman, 2003).

NAV2 encodes a member of the neuron navigator gene family, which mayplay a role in cellular growth and migration. NAV2 was shown to bespecifically expressed in a group of colon cancers and treatment ofcolon-cancer cells with antisense oligonucleotides for NAV2 inducedapoptosis (Ishiguro et al., 2002).

In liver cancer cells the loss of p53 has been shown to be responsiblefor NES expression and in breast cancer NES contributes to cancerdevelopment by enhancing Wnt/beta-catenin activation (Zhao et al., 2014;Tschaharganeh et al., 2014). Increased NES expression has been reportedin various tumor cells, including pancreatic ductal adenocarcinoma,malignant melanoma, uterine, prostate, breast and liver cancers. NESexpression correlates with aggressive features, metastasis and is abiomarker for poor prognosis. Furthermore, NES may be a marker for newlysynthesized tumor vessels and has also been suggested as a therapeutictarget to inhibit tumor angiogenesis (Ishiwata et al., 2011; Su et al.,2013; Matsuda et al., 2016; Hope et al., 2016).

NME5 is highly expressed in testis and some types of human cancer, likepancreatic cancer and breast cancer, and is associated with innateresistance to gemcitabine in pancreatic cancer cells (Parris et al.,2010; Li et al., 2012a; Li et al., 2012b).

NUP160-SLC43A3 is a recurrent fusion oncogene in angiosarcoma andassociated with tumor progression (Shimozono et al., 2015).

The P2RX7 system is an important pro-apoptosis modulator in epithelialcells and plays a role in chemoprevention in papillomas and epithelialcancers. Statins, cholesterol-lowering drugs, may reduce theinvasiveness and risk of aggressive prostate cancer via P2RX7. Also,P2X7 single-nucleotide polymorphisms could be exploited as diagnosticbiomarkers for the development of tailored therapies (Fu et al., 2009;Gorodeski, 2009; Ghalali et al., 2014; Roger et al., 2015; De et al.,2016). P2RX7 expression levels are elevated in primary bone tumors aswell as in other malignancies such as multiple myeloma, neuroblastoma,breast, and prostate cancer. There is evidence that P2RX7 triggersNFATc1, PI3K/Akt, ROCK, and VEGF pathways in osteoblasts promoting tumordevelopment (Adinolfi et al., 2012). P2RX7 is a potential prognosticmarker in hepatocellular carcinoma, where high peritumoral P2X7expression indicates unfavorable overall survival (Liu et al., 2015a).

PARVA is over-expressed in colorectal cancer, where it correlatessignificantly with tumor invasion, lymph node metastasis, and diseasestage, as well as with the over-expression of integrin-linked kinase,p-AKT, and nuclear β-catenin and the down-regulation of E-cadherin(Bravou et al., 2015). Over-expression of PARVA promoted tumorigenicity,angiogenesis and metastasis of lung adenocarcinoma by influencing ILKsignaling and a subsequent phosphorylation of Akt and GSK3beta (Huang etal., 2015). PARVA was frequently over-expressed in ovarian cancer,non-small cell lung carcinoma, prostate cancer and human hepatocellularcarcinoma, where its over-expression positively correlated with tumorsize, stage, and metastasis by enhancing survivin protein, β-catenin,and mammalian target of rapamycin pathways and suppressing p53 (Orr etal., 2012; Davidson et al., 2013; Augustin et al., 2013; Ng et al.,2013; Seydi et al., 2015). Furthermore, it was shown that PARVA isfrequently regulated by phosphorylation in breast cancer cells leadingto matrix degradation and cell invasion via regulation of Rho GTPasesignaling (Pignatelli et al., 2012). PARVA was found to be up-regulatedin prostate cancer and invasive lobular carcinoma being able to form anIPP complex with integrin-linked kinase and PINCH, that functions as asignaling platform for integrins (Kim et al., 2015b; Aakula et al.,2016; Ito et al., 2014).

PBK promotes lung cancer resistance to EGFR tyrosine kinase inhibitorsby phosphorylating and activating c-Jun (Li et al., 2016b).Over-expression of PBK confers malignant phenotype in prostate cancervia the regulation of E2F1 (Chen et al., 2015a). Targeting PBK decreasesgrowth and survival of glioma initiating cells in vitro and attenuatestumor growth in vivo (Joel et al., 2015). PBK inhibitor induces completetumor regression in xenograft models of human cancer through inhibitionof cytokinesis (Matsuo et al., 2014).

Elevated levels of PI4KA were observed in hepatocellular carcinomaversus normal liver tissue. In addition, the PI4KA gene was detected inpancreatic cancer cell line (Ishikawa et al., 2003; Ilboudo et al.,2014). Patients suffering from hepatocellular carcinoma with higherPI4KA mRNA concentrations had a higher risk of tumor recurrence as wellas shorter disease-specific survival (Ilboudo et al., 2014). Recently,PI4KA has been identified to be involved in cell proliferation andresistance to cisplatin treatment in a medulloblastoma cell line. Othershave revealed that PI4KA plays a crucial role in invasion and metastasisin pancreatic cancer (Ishikawa et al., 2003; Guerreiro et al., 2011).

PLA2G4A expression is up-regulated in colorectal cancer, bladdercarcinoma, which provides COX-2 with arachidonic acid, resulting inincreased prostaglandin levels. Up-regulation may occur due to prolongedinflammatory conditions (Osterstrom et al., 2002; Dong et al., 2005;Parhamifar et al., 2005; Shi et al., 2006). In gastric cancer increasedPLA2G4A and COX-2 expression were both associated with unfavorablesurvival and PLA2G4A might serve as a promising target for futuretherapeutic approaches to gastric cancer combined with COX-2 inhibitors.Also, inhibition of PLA2G4A may sensitize tumors to radiation therapy(Linkous et al., 2009; Zhang et al., 2013).

PLEC encodes the plakin family member plectin, a protein involved in thecross-linking and organization of the cytoskeleton and adhesioncomplexes (Bouameur et al., 2014). PLEC is over-expressed in colorectaladenocarcinoma, head and neck squamous cell carcinoma and pancreaticcancer (Lee et al., 2004; Katada et al., 2012; Bausch et al., 2011).

PMEL was described as a target for anti-body drug conjugate therapy inmelanoma (Chen et al., 2012). PMEL was shown to be associated withpaclitaxel and cisplatin resistance in melanoma (Hertzman et al., 2013).PMEL was described as one out of nine proteins applicable in a targetedselected reaction monitoring assay which provides potential advancementsin the diagnosis of malignant melanoma (Welinder et al., 2014a).

POLM is an error-prone DNA repair enzyme that is prone to inducetemplate/primer misalignments and mis-incorporation. High expressionlevels are thought to be involved in somatic hyper-mutation in aBurkitt's lymphoma-derived B cell line (Ruiz et al., 2004; Fernandez andAlbar, 2012).

Some researchers have observed a significant increase in PRKAR1Aexpression in undifferentiated thyroid carcinomas compared to normalthyroid tissue and differentiated thyroid tumors. On the contrary,down-regulation of PRKAR1A expression was reported in a subset ofodontogenic tumors. Another group revealed that PRKAR1A could beinvolved in the pathogenesis of odontogenic myxomas as well as insporadic adrenocortical adenomas (Bertherat et al., 2003; Perdigao etal., 2005; Ferrero et al., 2015; Sousa et al., 2015).

PSMA2 is differentially expressed in plasma cells of multiple myelomaand immunoglobulin light chain amyloidosis (Abraham et al., 2005). PSMA2is down-regulated in methotrexate-resistant breast cancer MCF-7 cells(Chen et al., 2014c).

PSMB7 expression is increased in most cancer types, along with otherconstitutive proteasome genes. In breast cancer and colorectal cancerhigh PSMB7 expression has been reported as an unfavorable prognosticmarker. In hepatocellular carcinoma and breast cancer it may contributeto chemotherapy resistance (Rho et al., 2008; Munkacsy et al., 2010; Tanet al., 2014; Rouette et al., 2016).

PTPN14 induces TGF-beta signaling, regulates endothelial-mesenchymaltransition, and organogenesis (Wyatt and Khew-Goodall, 2008). PTPN14 isdown-regulated in cholangiocarcinoma and is inversely correlated withclinical-pathological features and survival (Wang et al., 2015d; Wang etal., 2015c). PTPN14 inhibits trafficking of soluble and membrane-boundproteins, resulting in prevention of metastasis (Belle et al., 2015).PTPN14 negatively regulates the oncoprotein Yes-associated protein(YAP), a key protein in the Hippo pathway, which is responsible fororgan size and tumorigenesis (Liu et al., 2013; Huang et al., 2013; Linet al., 2013). Loss-of-function mutations in PTPN14 are involved inneuroblastoma relapse, breast cancer, and colorectal cancer (Laczmanskaand Sasiadek, 2011; Wang et al., 2004; Schramm et al., 2015; Wyatt andKhew-Goodall, 2008).

RAD21 is a component of the cohesin complex, crucial for chromosomesegregation and DNA repair. RAD21 is over-expressed in gastrointestinaltumors, colorectal carcinoma, advanced endometrial cancer, prostatecancer and breast cancer (Atienza et al., 2005; Deb et al., 2014; Porkkaet al., 2004; Supernat et al., 2012; Xu et al., 2014).

RAD50 forms the MRN complex with MRE11 and NBS1, a complex that binds toDNA and displays numerous enzymatic activities that are required fornon-homologous joining of DNA ends and is important for double-strandbreak repair, cell cycle checkpoint activation, telomere maintenance andmeiotic recombination. Mutations in this gene are the cause of Nijmegenbreakage syndrome-like disorder (RefSeq, 2002). RAD50 deletion appearsto be common in basal-like breast cancer and ovarian cancer and wasassociated with significantly better overall survival. Deletion oftenoccurs together with deletions of BRCA1, RB1, TP53, PTEN and INPP4B, andRAD50 and INPP4B expression levels have a synergistic influence onbreast cancer survival, possibly through their effects on treatmentresponse (Weigman et al., 2012; Dai et al., 2015; Zhang et al., 2016a).In colorectal cancer over-expression of RAD50 may be involved in cancerprogression. RAD50 becomes highly expressed if transcription factor BTF3is over-expressed and over-expression in primary tumors seems to berelated to early tumor stage, better differentiation, high inflammatoryinfiltration and p53 over-expression (Wang et al., 2013a; Gao et al.,2008). RAD50 has been found to be frequently mutated in hereditarybreast and ovarian cancer, colorectal cancer and in metastatic non-smallcell lung RAD50 mutation contributes to a curative response to systemiccombination therapy (Al-Ahmadie et al., 2014; Rajkumar et al., 2015).

A RANBP2-ALK gene fusion is detectable in different cancer entitiesincluding leukemias and lymphomas (Lim et al., 2014; Chen and Lee, 2008;Maesako et al., 2014; Lee et al., 2014). RANBP2 sumoylates Topo II alphain mitosis, and this modification is required for its properlocalization to inner centromeres. Thereby, RANBP2 plays an importantrole in preventing chromosome segregation errors (Navarro and Bachant,2008; Dawlaty et al., 2008).

Researchers have identified the RAPGEF6 as an upstream activator of Rap1required for the maturation of adherent junctions in the lung carcinomacells (Dube et al., 2008). Another group has demonstrated the formationof a complex between JAM-A, AF-6 and the RAPGEF6 in breast cancer cellsand in primary cultures from breast cancer patients (McSherry et al.,2011).

RBM4, a splicing factor over-expressed in several entities,alternatively splices RGPD1 (Markus et al., 2016). CG-1521, ananti-proliferative cancer drug, up-regulates RGPD1 expression(Chatterjee et al., 2013).

RBM4, a splicing factor over-expressed in several entities,alternatively splices RGPD2 (Markus et al., 2016). NEAT1-RGPD2,RGPD2-FASN, and RGPD2-MALAT1 are fusion transcripts detected in primarybreast cancer (Kim et al., 2015a). RGPD2 may be an ALK fusion partner inacute myelomonocytic leukemia (Lim et al., 2014). CG-1521, ananti-proliferative cancer drug, up-regulates RGPD2 expression(Chatterjee et al., 2013).

RGPD3 encodes RANBP2-like and GRIP domain containing 3 which is locatedin a cluster of Ran-binding protein related genes on chromosome 2 whicharose through duplication in primates. The encoded protein contains anN-terminal TPR (tetratricopeptide repeat) domain, two Ran-bindingdomains, and a C-terminal GRIP domain (golgin-97, RanBP2alpha, Imh1p andp230/golgin-245) domain (RefSeq, 2002). RGPD3 is a cancer gene with 3DHotMAPS regions in pancreatic adenocarcinoma (Tokheim et al., 2016).RGPD3 may be a target gene of HOXB7 (Heinonen et al., 2015). Dioscinalters RGPD3 expression in colon cancer cells (Chen et al., 2014a). Agene fusion transcript of ANAPC1 with RGPD3 has been reported innasopharyngeal carcinoma (Chung et al., 2013). CG-1521, ananti-proliferative cancer drug, up-regulates RGPD3 expression(Chatterjee et al., 2013). RGPD3 is mutated in gastrotintestinal stromaltumors and meningiomas (Brastianos et al., 2013).

RGPD8 is predominantly altered in prostate cancer and glioma (Meszaroset al., 2016). RGPD8 is part of a run of homozygosity associated withthyroid cancer (Thomsen et al., 2016). CG-1521, an anti-proliferativecancer drug, up-regulates RGPD8 expression (Chatterjee et al., 2013).

As RICTOR is able to interact with mTOR, it is playing a major role inthe PI3K/AKt/mTOR signaling pathway and was found to be up-regulated invarious cancer types such as small cell lung cancer, large-cellneuroendocrine carcinoma of the lung, breast cancer pancreatic cancerand colorectal cancer (Suh et al., 2016; Morrison et al., 2016; Miyoshiet al., 2016; Visuttijai et al., 2016; Sticz et al., 2016; Driscoll etal., 2016; Sakre et al., 2016). RICTOR polymorphisms were found innon-small cell lung cancer and breast cancer and were related to theprogression and metastasis of these cancers (Zhou et al., 2016; Wang etal., 2016b). RICTOR takes part in forming the PRICKLE1-MINK1-RICTORcomplex, which is required for activation of AKT, regulation of focaladhesions and cancer cell migration (Daulat et al., 2016). RICTORover-expression is associated with the carcinogenesis and progression ofcolorectal cancer and can be an independent indicator for evaluating theprognosis of colorectal cancer patients (Wang et al., 2016a).

ROPN1 is a cancer-testis antigen expressed in prostate cancer, acutemyeloid leukemia, multiple myeloma and basal like breast cancer and hasbeen suggested as a potential serological biomarker for prostate cancer.As a cancer-testis antigen it represents an attractive target for tumorimmunotherapy (Chiriva-Internati et al., 2011; Atanackovic et al., 2011;Ivanov et al., 2013; Adeola et al., 2016).

S100A1 was found to be down-regulated in oral cancer and bladder tumors,but up-regulated in ovarian cancer and in gastric cancer up-regulationof S100A1 was caused by over-expression of prion protein PRNP (Hibbs etal., 2004; Liang et al., 2007; Yao et al., 2007; Tyszkiewicz et al.,2014). S100A1 may be a potentially powerful marker to differentiatesubtypes of cancer. It can help distinguish chromophobe renal cellcarcinoma from renal oncocytoma and is up-regulated in basal-type breastcancers compared to non-basal types. S100A1 may also serve as a markerfor poor prognosis of endometrioid subtypes of cancer (Li et al., 2007;DeRycke et al., 2009; McKiernan et al., 2011).

SERPINE2 creates tumor-promoting conditions in the tumormicroenvironment and regulates tumor matrix deposition in multiple ways.It also is involved in vascular mimicry (Smirnova et al., 2016).SERPINE2 is over-expressed in breast cancer, prostate cancer andtesticular cancer and promotes the development of metastasis. In gastriccancer SERPINE2 up-regulation may contribute to the aggressive phenotypeand has been suggested as a novel prognostic factor and as an anticancertarget, e.g. through inhibition by monoclonal antibodies (Smirnova etal., 2016; Nagahara et al., 2010; Kousted et al., 2014; Wang et al.,2015b; Wagenblast et al., 2015). In prostate cancer SERPINE2 expressionappears to down-regulate distinct oncogenic pathways and inhibithedgehog-signaling and angiogenesis (McKee et al., 2013; McKee et al.,2015).

SGK1 expression is rapidly up-regulated by glucocorticoid administrationwhich may decrease chemotherapy effectiveness in ovarian cancer. Inturn, the isoflavinoid Genistein has been found to have an inhibitoryeffect on colorectal cancer by reducing SGK1 expression (Melhem et al.,2009; Qin et al., 2015). Increased SGK1 expression has been found inseveral human tumors, including prostate carcinoma, non-small cell lungcancer and hepatocellular carcinoma. SGK1 has anti-apoptotic propertiesand regulates cell survival, proliferation and differentiation viaphosphorylation of MDM2, which leads to the ubiquitination andproteasomal degradation of p53. Direct SGK1 inhibition can be effectivein hepatic cancer therapy, either alone or in combination withradiotherapy (Lang et al., 2010; Abbruzzese et al., 2012; Isikbay etal., 2014; Talarico et al., 2015).

SGK3 function was shown to be associated with the oncogenic driverINPP4B in colon cancer and in breast cancer (Gasser et al., 2014; Guo etal., 2015). SGK3 was described as a down-stream mediator ofphosphatidylinositol 3-kinase oncogenic signaling which mediates pivotalroles in oncogenic progress in various cancers, including breast cancer,ovarian cancer and hepatocellular carcinoma (Hou et al., 2015). SGK3 wasdescribed to serve as a hallmark interacting with numerous molecules incell proliferation, growth, migration and tumor angiogenesis (Hou etal., 2015). SGK3 was shown to promote hepatocellular carcinoma growthand survival through inactivating glycogen synthase kinase 3 beta andBcl-2-associated death promoter, respectively (Liu et al., 2012). SGK3was shown to be associated with poor outcome in hepatocellular carcinomapatients (Liu et al., 2012). Thus, SGK3 may provide a prognosticbiomarker for hepatocellular carcinoma outcome prediction and a noveltherapeutic target (Liu et al., 2012). SGK3 was described as animportant mediator of PDK1 activities in melanoma cells whichcontributes to the growth of BRAF-mutant melanomas and may be apotential therapeutic target (Scortegagna et al., 2015). SGK3 wasdescribed as an androgen receptor transcriptional target that promotesprostate cell proliferation through activation of p70 S6 kinase andup-regulation of cyclin D1 (Wang et al., 2014). Knock-down of SGK3 wasshown to decrease LNCaP prostate cancer cell proliferation by inhibitingG1 to S phase cell cycle progression (Wang et al., 2014). SGK3 was shownto be associated with estrogen receptor expression in breast cancer andits expression was shown to be positively correlated with tumorprognosis (Xu et al., 2012).

It was shown that SHC4 represents an EGFR-binding partner and Grb2platform and acts non-canonically to promote phosphorylation of selectEGFR residues (Wills et al., 2014). SHC4 interacts with membranereceptors, is involved in central cascades including MAPK and Akt, andis unconventionally contributed to oxidative stress and apoptosis (Willsand Jones, 2012).

Transcription levels of SLC4A5 were found to be significantly higher intherapy resistant ovary carcinoma cells (Pelzl et al., 2015). SLC4A5represents a pigmentation gene that is involved in phenotypic traitsincluding fair skin, light-colored eyes, and poor tanning ability, whichare all linked to melanoma risk (Nan et al., 2009; Pho and Leachman,2010).

SLC29A1 is a major transporter involved in gemcitabine and5-fluorouracil intracellular uptake in chemotherapy and it was found tobe up-regulated in gastric cancer and colorectal carcinoma. Inpancreatic cancer it has been validated as a predictive marker for thebenefit of gemcitabine therapy and has been suggested to be the same incholangiocarcinoma (Shimakata et al., 2016; Hagmann et al., 2010; Northet al., 2014; Nordh et al., 2014; Brandi et al., 2016; Kunicka et al.,2016). SLC29A1 has been identified as a marker to distinguish metastasesof clear cell renal cell carcinoma to the adrenal from primary adrenalcortical neoplasms or normal adrenal (Li et al., 2015a).

SLC45A2 was shown to be highly enriched in melanoma cell lines (Bin etal., 2015). Single nucleotide polymorphisms in SLC45A2 were associatedwith cutaneous melanoma risk, as well as cutaneous basal cell carcinomaand squamous cell carcinoma (Antonopoulou et al., 2015; Stacey et al.,2009).

SNCA is widely expressed in a variety of brain tumors, such asmedulloblastoma, neuroblastoma, pineoblastoma, and ganglioma and also inthe peripheral cancers, including ovarian cancer and breast cancer.Determining the levels of SNCA expression in tissues may be a biomarkerto detect metastatic melanoma (Fujita et al., 2007; Matsuo and Kamitani,2010; Welinder et al., 2014b). The SNCA promotor is frequentlyhyper-methylated in colorectal cancers and adenomas and might be asuitable biomarker for early non-invasive detection (Lind et al., 2011;Li et al., 2015e).

Knock-down of SNRPN in the Daoy human medulloblastoma cell line wasshown to reduce proliferation and colony formation ability, indicatingthat SNRPN may be a potential novel target for the development ofpharmacological therapeutics in human medulloblastoma (Jing et al.,2015). Knock-down of SNRPN in the BxPC-3 pancreatic adenocarcinoma cellline was shown to reduce the proliferation ability and impaired cellcolony formation. Its depletion was also shown to led to S phase cellcycle arrest and apoptosis (Ma et al., 2015). Depletion of SNRPN inBxPC-3 pancreatic adenocarcinoma cells was also shown to lead to S phasecell cycle arrest and apoptosis (Ma et al., 2015). Knock-down of SNRPNwas shown to result in a significant decrease in both invasion andproliferation in specifically Caucasian prostate cancer cell lines(Devaney et al., 2015).

SNX14 is down-regulated upon rasV12/E1A transformation of mouseembryonic fibroblasts and may be associated with tumor development(Vasseur et al., 2005).

SOX5 is up-regulated in breast cancer cells and hepatocellularcarcinoma. It induces epithelial to mesenchymal transition bytransactivation of Twist1 (Moon et al., 2014; Wang et al., 2015a). SOX5is expressed in glioma tissues, but not in normal adult tissues, exceptin testis. Additionally, antibodies against SOX5 were detected in serafrom 8 of 27 glioma patients and patients who showed IgG responsesagainst SOX5 exhibited significantly better survival periods thanpatients without SOX5 antibodies (Ueda et al., 2007). Together withother novel hypermethylated genes (AKR1B1, CHST10, ELOVL4, STK33,ZNF304) SOX5 was found as a potential methylation biomarker andtherapeutic target of vincristine in colorectal carcinoma (Pei et al.,2014).

SOX6 encodes a member of the D subfamily of sex determining regiony-related transcription factors that are characterized by a conservedDNA-binding and their ability to bind the minor groove of DNA. SOX6 is atranscriptional activator that is required for normal development of thecentral nervous system, chondrogenesis and maintenance of cardiac andskeletal muscle cells. It interacts with other family members tocooperatively activate gene expression (RefSeq, 2002). SOX6 functions asa tumor suppressor in myeloid leukemia, hepatocellular carcinoma andesophageal squamous cell carcinoma (ESCC). SOX6 was found to befrequently down-regulated in ESCC and down-regulation correlates withpoor survival. The tumor-suppressive mechanism of SOX6 was associatedwith its role in G1/S cell-cycle arrest by up-regulating expressions ofp53 and p21 and down-regulating expressions of cyclins (Qin et al.,2011b; Cantu et al., 2011; Guo et al., 2013). SOX-6 is considered acancer-testis gene and was found to be expressed in a high percentage ofhuman central nervous system tumors, including meningiomas andglioblastomas and could be the potential target of immunotherapy forcentral nervous system tumors (Lee et al., 2008).

SRGAP1 was shown to be associated with glioblastoma multiforme in thecell lines U87-IM3 and U251-IM3, familial forms of non-medullary thyroidcarcinoma, papillary thyroid carcinoma and epithelial ovarian cancer (Heet al., 2013; Chen et al., 2014b; Pereira et al., 2015; Koo et al.,2015).

SRGAP2 has been found to be up-regulated in an investigation of themolecular characteristics of recurrent triple-negative breast cancer andwas associated with cell adhesion and motility (Tsai et al., 2015).

SRGAP3 expression is down-regulated in several breast cancer cell linesand SRGAP3 exhibits has tumor suppressor-like activity in all mammaryepithelial cells, likely through its activity as a negative regulator ofRac1 (Lahoz and Hall, 2013). In pilocytic astrocytomas a tandemduplication at 3p25 was observed, which produces an in-frame oncogenicfusion between SRGAP3 and RAF1 hat may contribute to tumorigenesis(Jones et al., 2009).

The human ortholog of SSR4 was shown to be differentially expressed inthe opossum melanoma cell lines TD6b and TD15L2 and up-regulated intumors of advanced stages, implicating SSR4 as a candidate gene withpotential functions that might be associated with ultraviolet-inducedmelanomagenesis and metastasis (Wang and VandeBerg, 2004). The mRNAlevel of SSR4 was shown to be enriched in the osteosarcoma cell linesOHS, SaOS-2 and KPDXM compared to normal osteoblast cells (Olstad etal., 2003).

STAM has been found to be over-expressed in locally advanced cervicalcancer and in tumors in young patients with spinal ependymomas(Korshunov et al., 2003; Campos-Parra et al., 2016). STAM is adownstream target of ZNF331, a gene down-regulated in gastric cancer,which then leads to down-regulation of STAM as well (Yu et al., 2013).STAM has been associated with the unfavorable 11q deletion in chroniclymphocytic leukemia (Aalto et al., 2001).

STAT2 operates as a positive regulator in the transcriptional activationresponse elicited by IFNs (Steen and Gamero, 2012). STAT2 may regulatetumor cell response to interferons (Shodeinde et al., 2013). A linkbetween STAT2 and tumorigenesis was observed in transgenic mice lackingSTAT2 (Yue et al., 2015) or expressing constitutively IFN-α in the brain(Wang et al., 2003).

TANC1 was found to play a role in regenerating damaged muscle and issuggested to influence the development of late radiation-induced damagein prostate cancer patients (Fachal et al., 2014). Ectopic TANC1expression in rhabdomyosarcoma (RMS) causes misregulated myoblast fusionproteins, which might represent candidates for targeted RMS therapy(Avirneni-Vadlamudi et al., 2012).

Families presenting with Oral-Facial-Digital syndrome type 6 (OFD6) havelikely pathogenic mutations in TMEM17 causing ciliogenesis defects (Liet al., 2016a).

TMEM209 is widely expressed in lung cancer, in which it is localized tothe nuclear envelope, Golgi apparatus, and the cytoplasm of lung cancercells. Ectopic over-expression of TMEM209 promoted cell growth, whereasTMEM209 attenuation was sufficient to block growth (Fujitomo et al.,2012).

It was shown that TSPAN14 is significantly up-regulated in cancer cellstreated with coumarin- and benzimidazole-containing compounds, whichpossess anti-tumor activity by inducing caspase-dependent apoptosis (Liuet al., 2015b). TSPAN14 was found to be up-regulated in grade 1 lungtumors, suggesting that structural changes of these genes could play arole in cancer promotion (Bankovic et al., 2010).

UTP20 expression is decreased in metastatic human breast tumor celllines (Schwirzke et al., 1998; Goodison et al., 2003). UTP20 isexpressed at high levels in gastric cancer tissues and premalignantlesions implicating the involvement of UTP20 in cell transformation(Xing et al., 2005).

VGLL4 acts as a tumor suppressor in gastric cancer, lung cancer andesophageal squamous cell carcinoma by negatively regulating the YAP-TEADtranscriptional complex and inhibiting YAP induced tumorigenesis. VGLL4has been shown to be down-regulated during the progression of gastriccancer and esophageal squamous carcinoma (Zhang et al., 2014c; Jiao etal., 2014; Jiang et al., 2015; Li et al., 2015c). VGLL4 may also inhibitepithelial-mesenchymal transition in gastric cancer through the Wnt/betasignaling pathway (Li et al., 2015b).

WDFY3 was shown to be down-regulated in colorectal cancer (Piepoli etal., 2012).

It was shown that WDR35 is one of the key genes for chronic myeloidleukemia progression and is differentially methylated in acutelymphoblastic leukemia (Nordlund et al., 2012; Zhang et al., 2014b).WDR35 regulates cilium assembly by selectively regulating transport ofdistinct cargoes, is essential for the entry of many membrane proteinsinto the cilium and is mutated in several cargo transport mediateddiseases (Fu et al., 2016). WDR35 expression is regulated by theCaMKK/AMPK/p38 MAPK pathway as well as by NF-kappaB (Harato et al.,2012; Huang et al., 2014b; Huang et al., 2014a).

WDR6 inhibits the colony formation of cervical cancer cells viaregulation of the LKB1 pathway and stimulation of p27 promoter activity(Xie et al., 2007). WDR6 plays an important role in hepatocarcinogenesisand can be used as a detection marker of hepatocellular proliferativelesions (Yafune et al., 2013).

WDR7 expression is de-regulated by copy number alterations in gastriccancer and shows an elevated expression in numerous malignant cell lines(Junnila et al., 2010; Sanders et al., 2000).

ZBTB3 may play a critical role in cancer cell growth in human melanoma,lung carcinoma, and breast carcinoma via the ROS detoxification system(Lim, 2014). Suppression of ZBTB3 activates a caspase cascade, includingcaspase-9, -3, and PARP leading to cellular apoptosis and mighttherefore represent a potential target for selective cancer treatments(Lim, 2014).

ZMYM1 is a major interactor of ZNF131 which acts in estrogen signalingand breast cancer proliferation (Oh and Chung, 2012; Kim et al., 2016).

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, or 13 or longer, andin case of MHC class II peptides (elongated variants of the peptides ofthe invention) they can be as long as 14, 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 5 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 − (1 −Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype Allele Population fromallele frequency A*02 Caucasian (North America) 49.1% A*02 AfricanAmerican (North America) 34.1% A*02 Asian American (North America) 43.2%A*02 Latin American (North American) 48.3% DR1 Caucasian (North America)19.4% DR2 Caucasian (North America) 28.2% DR3 Caucasian (North America)20.6% DR4 Caucasian (North America) 30.7% DR5 Caucasian (North America)23.3% DR6 Caucasian (North America) 26.7% DR7 Caucasian (North America)24.8% DR8 Caucasian (North America)  5.7% DR9 Caucasian (North America) 2.1% DR1 African (North) American 13.20%  DR2 African (North) American29.80%  DR3 African (North) American 24.80%  DR4 African (North)American 11.10%  DR5 African (North) American 31.10%  DR6 African(North) American 33.70%  DR7 African (North) American 19.20%  DR8African (North) American 12.10%  DR9 African (North) American 5.80% DR1Asian (North) American 6.80% DR2 Asian (North) American 33.80%  DR3Asian (North) American 9.20% DR4 Asian (North) American 28.60%  DR5Asian (North) American 30.00%  DR6 Asian (North) American 25.10%  DR7Asian (North) American 13.40%  DR8 Asian (North) American 12.70%  DR9Asian (North) American 18.60%  DR1 Latin (North) American 15.30%  DR2Latin (North) American 21.20%  DR3 Latin (North) American 15.20%  DR4Latin (North) American 36.80%  DR5 Latin (North) American 20.00%  DR6Latin (North) American 31.10%  DR7 Latin (North) American 20.20%  DR8Latin (North) American 18.60%  DR9 Latin (North) American 2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that are A*02positive, whereas no selection for MHC class II allotypes is necessarydue to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iiii) the alignment has to start at position 1 of the alignedsequences;and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 237 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 237, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 237. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 237, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or—II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or —II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acid whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptides according to SEQ ID NO: 1,10, and 20 Position 1 2 3 4 5 6 7 8 9 10 SEQ ID No 1 F L D V K E L M LVariant V I A M V M I M M A A V A I A A A V V V I V V A T V T I T T A QV Q I Q Q A SEQ ID No K M T Q Y I T E L 10 Variant L V L I L L A V I A AV A I A A A V V V I V V A T V T I T T A Q V Q I Q Q A SEQ ID No V I S PH G I A S V 20 Variant L L I L L L A M M I M L M A A A I A L A A V V I VL V A T T I T L T A Q Q I Q L Q A

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 237.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 237 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidation,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich (www.sigma-aldrich.com)provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID No: 1 to SEQ ID No: 237and has been synthetically produced (e.g. synthesized) as apharmaceutically acceptable salt. Methods to synthetically producepeptides are well known in the art. The salts of the peptides accordingto the present invention differ substantially from the peptides in theirstate(s) in vivo, as the peptides as generated in vivo are no salts. Thenon-natural salt form of the peptide mediates the solubility of thepeptide, in particular in the context of pharmaceutical compositionscomprising the peptides, e.g. the peptide vaccines as disclosed herein.A sufficient and at least substantial solubility of the peptide(s) isrequired in order to efficiently provide the peptides to the subject tobe treated. Preferably, the salts are pharmaceutically acceptable saltsof the peptides. These salts according to the invention include alkalineand earth alkaline salts such as salts of the Hofmeister seriescomprising as anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻,I⁻, SCN⁻ and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺,Mn²⁺, Cu²⁺ and Ba²⁺. Particularly salts are selected from (NH₄)₃PO₄,(NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃,NH₄ClO₄, NH₄I, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO,Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄,K₂SO₄, KCH₃COO, KCl, KBr, KNO₃, KCIO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄,NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI, NaSCN, ZnCl₂Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CsClO₄,CSI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCI, LiBr,LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂,Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂,MnCl₂, Ca₃(PO₄)Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂, CaBr₂,Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂,BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, BaI₂, andBa(SCN)₂. Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄,KCl, NaCl, and CaCl₂, such as, for example, the chloride or acetate(trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981) and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoroacetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995) (cf. Example 1, FIGS. 1A to 1J).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from melanoma samples (N=18A*02-positive samples) with the fragmentation patterns of correspondingsynthetic reference peptides of identical sequences. Since the peptideswere directly identified as ligands of HLA molecules of primary tumors,these results provide direct evidence for the natural processing andpresentation of the identified peptides on primary cancer tissueobtained from 18 melanoma patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from melanoma tissue samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (seeexamples). All TUMAPs contained in the present application wereidentified with this approach on primary melanoma samples confirmingtheir presentation on primary melanoma.

TUMAPs identified on multiple melanoma and normal tissues werequantified using ion-counting of label-free LC-MS data. The methodassumes that LC-MS signal areas of a peptide correlate with itsabundance in the sample. All quantitative signals of a peptide invarious LC-MS experiments were normalized based on central tendency,averaged per sample and merged into a bar plot, called presentationprofile. The presentation profile consolidates different analysismethods like protein database search, spectral clustering, charge statedeconvolution (decharging) and retention time alignment andnormalization.

Besides over-presentation of the peptide, mRNA expression of theunderlying gene was tested. mRNA data were obtained via RNASeq analysesof normal tissues and cancer tissues (cf. Example 2, FIGS. 2A-2C). Anadditional source of normal tissue data was a database of publiclyavailable RNA expression data from around 3000 normal tissue samples(Lonsdale, 2013). Peptides which are derived from proteins whose codingmRNA is highly expressed in cancer tissue, but very low or absent invital normal tissues, were preferably included in the present invention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably melanoma that over- or exclusively presentthe peptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanmelanoma samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy skin cells or other normal tissue cells, demonstrating a highdegree of tumor association of the source genes (see Example 2).Moreover, the peptides themselves are strongly over-presented on tumortissue—“tumor tissue” in relation to this invention shall mean a samplefrom a patient suffering from melanoma, but not on normal tissues (seeExample 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. melanoma cells presenting the derivedpeptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides capable of binding to TCRs and antibodies whenpresented by an MHC molecule. The present description also relates tonucleic acids, vectors and host cells for expressing TCRs and peptidesof the present description; and methods of using the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to a peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta hetero-dimeric TCRs of the present description may have aTRAC constant domain sequence and a TRBC1 or TRBC2 constant domainsequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2constant domain sequence of the TCR may be linked by the nativedisulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 ofTRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, an peptide-HLA molecule complex, which is atleast double that of a TCR comprising the unmutated TCR alpha chainand/or unmutated TCR beta chain. Affinity-enhancement of tumor-specificTCRs, and its exploitation, relies on the existence of a window foroptimal TCR affinities. The existence of such a window is based onobservations that TCRs specific for HLA-A2-restricted pathogens have KDvalues that are generally about 10-fold lower when compared to TCRsspecific for HLA-A2-restricted tumor-associated self-antigens. It is nowknown, although tumor antigens have the potential to be immunogenic,because tumors arise from the individual's own cells only mutatedproteins or proteins with altered translational processing will be seenas foreign by the immune system. Antigens that are upregulated oroverexpressed (so called self-antigens) will not necessarily induce afunctional immune response against the tumor: T-cells expressing TCRsthat are highly reactive to these antigens will have been negativelyselected within the thymus in a process known as central tolerance,meaning that only T-cells with low-affinity TCRs for self-antigensremain. Therefore, affinity of TCRs or variants of the presentdescription to the peptides according to the invention can be enhancedby methods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with peptide of interest, incubating PBMCs obtained from thetransgenic mice with tetramer-phycoerythrin (PE), and isolating the highavidity T-cells by fluorescence activated cell sorting (FACS)-Caliburanalysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient. In anotheraspect, to obtain T-cells expressing TCRs of the present description,TCR RNAs are synthesized by techniques known in the art, e.g., in vitrotranscription systems. The in vitro-synthesized TCR RNAs are thenintroduced into primary CD8+ T-cells obtained from healthy donors byelectroporation to re-express tumor specific TCR-alpha and/or TCR-betachains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed. In addition to strong promoters,

TCR expression cassettes of the present description may containadditional elements that can enhance transgene expression, including acentral polypurine tract (cPPT), which promotes the nucleartranslocation of lentiviral constructs (Follenzi et al., 2000), and thewoodchuck hepatitis virus posttranscriptional regulatory element (wPRE),which increases the level of transgene expression by increasing RNAstability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced (Schmitt et al., 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “optimal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3 fusion) (Schmitt et al., 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 237, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore, differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example, afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed, aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 237,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 237, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 237 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:237 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 237, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 237.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of melanoma.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 237 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are melanoma cells or other solidor hematological tumor cells such as acute myelogenous leukemia, breastcancer, bile duct cancer, brain cancer, chronic lymphocytic leukemia,colorectal carcinoma, esophageal cancer, gallbladder cancer, gastriccancer, hepatocellular cancer, non-Hodgkin lymphoma, non-small cell lungcancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cellcancer, small cell lung cancer, urinary bladder cancer and uterinecancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of melanoma. The present invention also relates to the use ofthese novel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a melanoma marker (poly)peptide,delivery of a toxin to a melanoma cell expressing a cancer marker geneat an increased level, and/or inhibiting the activity of a melanomamarker polypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length melanoma marker polypeptides or fragmentsthereof may be used to generate the antibodies of the invention. Apolypeptide to be used for generating an antibody of the invention maybe partially or fully purified from a natural source, or may be producedusing recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 237polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the melanoma marker polypeptide usedto generate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating melanoma, theefficacy of the therapeutic antibody can be assessed in various wayswell known to the skilled practitioner. For instance, the size, number,and/or distribution of cancer in a subject receiving treatment may bemonitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occur in the absence ofantibody administration, is an efficacious antibody for treatment ofcancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S)) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably, the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 237, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively eliciting high-or low-avidity antigen-specific T cell responses with high efficiencyfrom blood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore, such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition, plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 237.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut in the tumor it is expressed. By “over-expressed” the inventors meanthat the polypeptide is present at a level at least 1.2-fold of thatpresent in normal tissue; preferably at least 2-fold, and morepreferably at least 5-fold or 10-fold the level present in normaltissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore, anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from melanoma, themedicament of the invention is preferably used to treat melanoma.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue of melanomapatients with various HLA-A HLA-B and HLA-C alleles. It may contain MHCclass I and MHC class II peptides or elongated MHC class I peptides. Inaddition to the tumor associated peptides collected from severalmelanoma tissues, the warehouse may contain HLA-A*02 and HLA-A*24 markerpeptides. These peptides allow comparison of the magnitude of T-cellimmunity induced by TUMAPS in a quantitative manner and hence allowimportant conclusion to be drawn on the capacity of the vaccine toelicit anti-tumor responses. Secondly, they function as importantpositive control peptides derived from a “non-self” antigen in the casethat any vaccine-induced T-cell responses to TUMAPs derived from “self”antigens in a patient are not observed. And thirdly, it may allowconclusions to be drawn, regarding the status of immunocompetence of thepatient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, melanoma samples from patientsand blood from healthy donors were analyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue (melanoma)compared with a range of normal organs and tissues3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as frommelanoma patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients' tumor and,where possible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (al) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (al) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from melanoma cells and since it was determined thatthese peptides are not or at lower levels present in normal tissues,these peptides can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for melanoma. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

FIGS. 1A through 1J show the over-presentation of various peptides innormal tissues (white bars) and melanoma (black bars). FIG. 1A) Genesymbol: S100A1, Peptide: FLDVKELML (SEQ ID NO.: 1), Tissues from left toright: 4 adipose tissues, 5 adrenal glands, 24 blood cells, 15 bloodvessels, 10 bone marrows, 14 brains, 7 breasts, 7 esophagi, 2 eyes, 3gallbladders, 16 hearts, 17 kidneys, 20 large intestines, 23 livers, 49lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 8 pancreases, 6 parathyroidglands, 1 peritoneum, 5 pituitary glands, 7 placentas, 1 pleura, 3prostates, 7 salivary glands, 5 skeletal muscles, 3 small intestines, 12spleens, 5 stomachs, 5 testes, 2 thymi, 2 thyroid glands, 11 tracheas, 7ureters, 8 urinary bladders, 6 uteri, 12 skins, 18 melanoma. FIG. 1B)Gene symbol: EXTL1, Peptide: VLFKDPVSV (SEQ ID NO.:3), Tissues from leftto right: 4 adipose tissues, 5 adrenal glands, 24 blood cells, 15 bloodvessels, 10 bone marrows, 14 brains, 7 breasts, 7 esophagi, 2 eyes, 3gallbladders, 16 hearts, 17 kidneys, 20 large intestines, 23 livers, 49lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 8 pancreases, 6 parathyroidglands, 1 peritoneum, 5 pituitary glands, 7 placentas, 1 pleura, 3prostates, 7 salivary glands, 5 skeletal muscles, 3 small intestines, 12spleens, 5 stomachs, 5 testes, 2 thymi, 2 thyroid glands, 11 tracheas, 7ureters, 8 urinary bladders, 6 uteri, 12 skins, 18 melanoma. FIG. 1C)Gene symbol: HMCN1, Peptide: IQSETTVTV (SEQ ID NO.: 13), Tissues fromleft to right: 4 adipose tissues, 5 adrenal glands, 24 blood cells, 15blood vessels, 10 bone marrows, 14 brains, 7 breasts, 7 esophagi, 2eyes, 3 gallbladders, 16 hearts, 17 kidneys, 20 large intestines, 23livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 8 pancreases, 6parathyroid glands, 1 peritoneum, 5 pituitary glands, 7 placentas, 1pleura, 3 prostates, 7 salivary glands, 5 skeletal muscles, 3 smallintestines, 12 spleens, 5 stomachs, 5 testes, 2 thymi, 2 thyroid glands,11 tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 12 skins, 18melanoma. FIG. 1D) Gene symbol: TMEM17, Peptide: NLQEKVPEL (SEQ ID NO.:7), Samples from left to right: 14 cancer tissues (1 brain cancer, 1breast cancer, 1 head-and-neck cancer, 3 lung cancers, 1 myeloid cellscancer, 1 ovarian cancer, 1 pancreas cancer, 4 melanomas, 1 uteruscancer). FIG. 1E) through 1J) show the over-presentation of variouspeptides in different cancer tissues (black dots). Upper part: Median MSsignal intensities from technical replicate measurements are plotted asdots for single HLA-A*02 positive normal (grey dots) and tumor samples(black dots) on which the peptide was detected. Tumor and normal samplesare grouped according to organ of origin, and box-and-whisker plotsrepresent median, 25th and 75th percentile (box), and minimum andmaximum (whiskers) of normalized signal intensities over multiplesamples. Normal organs are ordered according to risk categories (bloodcells, blood vessels, brain, liver, lung: high risk, grey dots;reproductive organs, breast, prostate: low risk, grey dots; all otherorgans: medium risk; grey dots). Lower part: The relative peptidedetection frequency in every organ is shown as spine plot. Numbers belowthe panel indicate number of samples on which the peptide was detectedout of the total number of samples analyzed for each organ (N=526 fornormal samples, N=562 for tumor samples). If the peptide has beendetected on a sample but could not be quantified for technical reasons,the sample is included in this representation of detection frequency,but no dot is shown in the upper part of the figure. Tissues (from leftto right): Normal samples: blood cells; bloodvess (blood vessels);brain; heart; liver; lung; adipose (adipose tissue); adren.gl. (adrenalgland); bile duct; bladder; BM (bone marrow); cartilage; esoph(esophagus); eye; gallb (gallbladder); head&neck; kidney; large_int(large intestine); LN (lymph node); nerve; pancreas; parathyr(parathyroid gland); perit (peritoneum); pituit (pituitary); pleura;skel.mus (skeletal muscle); skin; small_int (small intestine); spleen;stomach; thyroid; trachea; ureter; breast; ovary; placenta; prostate;testis; thymus; uterus. Tumor samples: AML: acute myeloid leukemia;BRCA: breast cancer; CCC: cholangiocellular carcinoma; CLL: chroniclymphocytic leukemia; CRC: colorectal cancer; GBC: gallbladder cancer;GBM: glioblastoma; GC: gastric cancer; GEJC: stomach cardia esophagus,cancer; HCC: hepatocellular carcinoma; HNSCC: head-and-neck cancer; MEL:melanoma; NHL: non-hodgkin lymphoma; NSCLC: non-small cell lung cancer;OC: ovarian cancer; OSCAR: esophageal cancer; PACA: pancreatic cancer;PRCA: prostate cancer; RCC: renal cell carcinoma; SCLC: small cell lungcancer; UBC: urinary bladder carcinoma; UEC: uterine and endometrialcancer. FIG. 1E) Gene symbols: HLA-B, HLA-C, Peptide: VLAVLGAVVAV (SEQID NO.: 19), FIG. 1F) Gene symbol: PARVA, Peptide: SLVAILHLL (SEQ IDNO.: 24), FIG. 1G) Gene symbol: METAP2, Peptide: TMIEICEKL (SEQ ID NO.:118), FIG. 1H) Gene symbol: UTP20, Peptide: QLMEGKVVL (SEQ ID NO.: 120),FIG. 1I) Gene symbol: SNRPN, Peptide: FLGEPASYLYL (SEQ ID NO.: 151),FIG. 1J) Gene symbol: IPO9, Peptide: SILDGLIHL (SEQ ID NO.: 209).

FIGS. 2A through 2C) show exemplary expression profiles of source genesof the present invention that are highly over-expressed or exclusivelyexpressed in melanoma in a panel of normal tissues (white bars) and 10melanoma samples (black bars). Tissues from left to right: 6 arteries, 2blood cells, 2 brains, 1 heart, 2 livers, 3 lungs, 2 veins, 1 adiposetissue, 1 adrenal gland, 5 bone marrows, 1 cartilage, 1 colon, 1esophagus, 2 eyes, 2 gallbladders, 2 head-and-neck and salivary glands,1 kidney, 6 lymph nodes, 4 pancreases, 2 peripheral nerves, 2 pituitaryglands, 1 rectum, 2 skeletal muscles, 1 skin, 1 small intestine, 1spleen, 1 stomach, 1 thyroid gland, 7 tracheas, 1 urinary bladder, 1breast, 5 ovaries, 5 placentas, 1 prostate, 1 testis, 1 thymus, 1uterus, 10 melanoma. FIG. 2A) Gene symbol: SLC24A5, FIG. 2B) Genesymbol: SLC45A2, FIG. 2C) Gene symbol: FMN1.

FIGS. 3A and 3B shows exemplary immunogenicity data: flow cytometryresults after peptide-specific multimer staining.

FIGS. 4A through 4C show exemplary results of peptide-specific in vitroCD8+ T cell responses of a healthy HLA-A*02+ donor. CD8+ T cells wereprimed using artificial APCs coated with anti-CD28 mAb and HLA-A*02 incomplex with SeqID No 8 peptide (FIG. 4A, left panel), SeqID No 12peptide (FIG. 4B, left panel) and SeqID No 155 peptide (FIG. 4C, leftpanel), respectively. After three cycles of stimulation, the detectionof peptide-reactive cells was performed by 2D multimer staining withA*02/SeqID No 8 (FIG. 4A), A*02/SeqID No 12 (FIG. 4B) or A*02/SeqID No155 (FIG. 4C). Right panels (FIGS. 4A, 4B and 4C) show control stainingof cells stimulated with irrelevant A*02/peptide complexes. Viablesinglet cells were gated for CD8+ lymphocytes. Boolean gates helpedexcluding false-positive events detected with multimers specific fordifferent peptides. Frequencies of specific multimer+ cells among CD8+lymphocytes are indicated.

EXAMPLES Example 1 Identification and Quantitation of Tumor AssociatedPeptides Presented on the Cell Surface Tissue Samples

Patients' tumor tissues were obtained from: Asterand (Detroit, Mich.,USA & Royston, Herts, UK); ProteoGenex Inc. (Culver City, Calif., USA),Tissue Solutions Ltd (Glasgow, UK); University Hospital Heidelberg(Heidelberg, Germany); and University Hospital Tübingen (Tübingen,Germany).

Normal tissues were obtained from Asterand (Detroit, Mich., USA &Royston, Herts, UK); Bio-Options Inc. (Brea, Calif., USA); BioServe(Beltsville, Md., USA); Capital BioScience Inc. (Rockville, Md., USA);Geneticist Inc. (Glendale, Calif., USA); Kyoto Prefectural University ofMedicine (KPUM) (Kyoto, Japan); ProteoGenex Inc. (Culver City, Calif.,USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Geneva(Geneva, Switzerland); University Hospital Heidelberg (Heidelberg,Germany); University Hospital Munich (Munich, Germany); and UniversityHospital Tübingen (Tübingen, Germany).

Written informed consents of all patients had been given before surgeryor autopsy. Tissues were shock-frozen immediately after excision andstored until isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, —B, C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOP5 strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the Orbitrap(R=30 000), which was followed by MS/MS scans also in the Orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose melanoma samples to a baseline of normal tissuesamples. Presentation profiles of exemplary over-presented peptides areshown in FIGS. 1A-1J. Presentation scores for exemplary peptides areshown in Table 8.

TABLE 8 Presentation scores. The table lists peptides that are veryhighly over-presented on tumors compared to a panel of normal tissues(+++), highly over-presented on tumors compared to a panel of normaltissues (++) or over-presented on tumors compared to a panel of normaltissues (+). The panel of normal tissues considered relevant forcomparison with tumors consisted of: adipose tissue, adrenal gland,blood cells, blood vessel bone marrow, brain, esophagus, eye,gallbladder, heart, kidney, large intestine, liver, lung, lymph node,nerve, pancreas, parathyroid gland, peritoneum, pituitary, pleura,salivary gland, skeletal muscle, skin, small intestine, spleen, stomach,thymus, thyroid gland, trachea, ureter, urinary bladder. SEQ ID PeptideNo Sequence Presentation 1 FLDVKELML +++ 2 VLLGENVEL +++ 3 VLFKDPVSV +++4 KTWDQVPFSV +++ 5 ILDEGHILQL +++ 6 SIPDTIASV +++ 7 NLQEKVPEL +++ 8SIIPYLLEA +++ 9 SLAGLVLYV +++ 10 KMTQYITEL +++ 11 TLIELLLPKL +++ 12RLDDKTTNV ++ 13 IQSETTVTV ++ 14 VLYEMLYGL +++ 17 GVVHGVATV ++ 18SLADVVDTL + 19 VLAVLGAVVAV +++ 20 VISPHGIASV +++ 21 FMYNFQLVTL ++ 22KLLELQELVL ++ 24 SLVAILHLL ++ 26 KIEDLIKYL +++ 27 TLWYVPLSL ++ 28IVDNTTMQL + 30 VLFPMDLAL +++ 31 FLPRKFPSL ++ 32 GLDIITNKV ++ 33SLYSYFQKV +++ 34 YLINFEIRSL +++ 35 ALFAAGANV +++ 36 SVNGFISTL +++ 37TLKEYLESL +++ 38 KLGFGTGVNVYL +++ 39 ALPPPPASI +++ 40 LLSNTVSTL +++ 41LLDDPTNAHFI +++ 42 VLKADVVLL +++ 43 LLPDPLYSL +++ 44 FLYTYIAKV +++ 45FVYGEPREL +++ 46 VMSSTLYTV +++ 47 ALDSDPVGL +++ 48 HLIGWTAFL +++ 49ALLSQDFEL +++ 50 HLDQIFQNL +++ 51 LIDKIIEYL +++ 52 NLDYAILKL +++ 53ILDEEKFNV +++ 54 LLDSGAFHL +++ 55 NLDKLYHGL +++ 56 ILDELVKSL +++ 57GILSFLPVL +++ 58 ILGDWSIQV +++ 59 IIDDVMKEL ++ 60 ILPEAQDYFL +++ 61KLSVHVTAL +++ 62 LLDTTQKYL +++ 63 SIDDSDPIV +++ 64 SLGPIMLTKI +++ 65TTLGGFAKV +++ 66 VMFEYGMRL +++ 67 YVDSEGIVRM +++ 68 FLAEAARSL +++ 69IIDDKPIGL +++ 70 LIDEAAQML +++ 71 SLDEVAVSL +++ 72 TLLEVDAIVNA +++ 73ELDKIYETL +++ 74 GTIPLIESL +++ 75 FMYAGQLTL +++ 76 QIDSIHLLL +++ 77SIDDVVKKL +++ 78 ALKDLVNLI +++ 79 AVDNILLKL +++ 80 FADELSHLL +++ 81FLDDGNQML +++ 82 GIDDLHISL +++ 83 GLDKVITVL +++ 84 GLDTILQNL +++ 86HTLPHEIVVNL +++ 87 IIDPPLHGQLL +++ 88 ILDGIIREL ++ 89 ILDNSPAFL +++ 90ILDYIHNGL +++ 91 ILLDRLFSV +++ 92 KLPGFPTQDDEV +++ 93 LLAKAVQNV +++ 94LLDAFSIKL +++ 95 LLDALQHEL +++ 96 LLDMSLVKL +++ 97 NLDATVTAL +++ 98NLPNTNSILGV +++ 99 NLPSELPQL +++ 100 NLREILQNV +++ 101 NVDENVAEL +++ 102RLPDQFSKL +++ 103 SLDAVMPHL +++ 104 SLDQIIQHL +++ 105 SLKQTVVTL +++ 106TLSEICEFI +++ 107 TLVAFLQQV +++ 108 TVIRPLPGL +++ 109 VIDDLIQKL +++ 110VLDTLTKVL +++ 111 VLDVSFNRL +++ 112 VLPAVLTRL +++ 113 VLYSLVSKI +++ 114VVDDIVSKL +++ 115 YIDDVFMGL +++ 116 LMDETMKEL ++ 117 KQQASQVLV ++ 118TMIEICEKL ++ 119 SLGLGFISRV ++ 120 QLMEGKVVL ++ 121 FLEDLVPYL + 122YVDDFGVSV ++ 125 YLFAFLNHL +++ 126 SLIDFVVTC + 127 TLISDIEAVKA +++ 129VLPDDLSGV + 130 GLVDVLYTA + 131 FVDPNGKISL ++ 132 FLDASGAKL + 133ALDPAYTTL ++ 134 LLDEVLHTM +++ 135 FLDDQETRL + 136 FAYDGKDYIAL ++ 137ILPSNLLTV + 138 YLDKTFYNL + 139 AVDATVNQV + 140 RLEAYLARV + 146GVGPVPARA + 149 YLDTFALKL + 155 GLAGFFASV ++ 156 ALMDTDGSGKLNL + 157HLFETISQA ++ 159 TILATVPLV ++ 160 ALDDISESI + 163 RLMANPEALKI ++ 164ALFFQLVDV ++ 165 ALIEVLQPLI ++ 166 SIIPPLFTV ++ 168 KLLAATLLL + 169TLLESIQHV + 170 KLKEAVEAI ++ 171 KVSGVILSV ++ 172 FLPAGIVAV ++ 173ALDDIIYRA + 175 VLDSVDVRL + 177 ILWDTLLRL + 178 FAYDGKDYIA +++ 179ALDDTVLQV + 180 KLAEALYIA + 181 GLIDLEANYL + 182 SVALVIHNV + 184VLFSSPPVILL + 187 SLPRPTPQA + 188 VVVDPIQSV +++ 189 KALQFLEEV +++ 190RLVSLITLL +++ 191 YLDKMNNNI +++ 192 KLFTQIFGV +++ 193 ALDEPTTNL ++ 194TLDDIMAAV ++ 195 IAAGIFNDL +++ 196 ALEPIDITV +++ 197 ALDSGFNSV + 198EVVDKINQV +++ 200 LLEEINHFL +++ 201 SLIDRTIKM +++ 202 RVAFKINSV +++ 203FLNEDISKL +++ 204 RMDEEFTKI +++ 205 SLKSKVLSV +++ 206 LLYEDIPDKV + 207VQIGDIVTV + 208 YSDDIPHAL ++ 209 SILDGLIHL ++ 211 FLPFLTTEV + 212LLKDSIVQL + 213 LLDPTNVFI + 214 VLMEMSYRL + 215 EVISKLYAV + 216TLLHFLAEL ++ 217 NMMSGISSV ++ 218 STLHLVLRL + 221 SLLPTEQPRL ++ 223FLETNVPLL + 224 ILDEPTNHL + 225 VLFGAVITGA + 226 VLNEYFHNV + 227FLLEQEKTQAL + 228 FLNLFNHTL + 229 LLEPFVHQV ++ 230 HLDEARTLL + 231KMVGDVTGA + 233 QLYNQIIKL + 235 ALADLQEAV ++

Example 2 Expression Profiling of Genes Encoding the Peptides of theInvention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA & Royston, Herts, UK); BioCat GmbH(Heidelberg, Germany); BioServe (Beltsville, Md., USA), CapitalBioScience Inc. (Rockville, Md., USA); Geneticist Inc. (Glendale,Calif., USA), Istituto Nazionale Tumori “Pascale” (Naples, Italy);ProteoGenex Inc. (Culver City, Calif., USA), and University HospitalHeidelberg (Heidelberg, Germany).

Total RNA from tumor tissues for RNASeq experiments was obtained from:Asterand (Detroit, Mich., USA & Royston, Herts, UK); ProteoGenex Inc.(Culver City, Calif., USA); Tissue Solutions Ltd (Glasgow, UK), andUniversity Hospital Tübingen (Tübingen, Germany).

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

RNAseq Experiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (IlluminaInc., San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolar and sequenced on the Illumina HiSeq2500 sequencer according to the manufacturer's instructions, generating50 bp single end reads. Processed reads are mapped to the human genome(GRCh38) using the STAR software. Expression data are provided ontranscript level as RPKM (Reads Per Kilobase per Million mapped reads,generated by the software Cufflinks) and on exon level (total reads,generated by the software Bedtools), based on annotations of the ensemblsequence database (Ensembl77). Exon reads are normalized for exon lengthand alignment size to obtain RPKM values.

Exemplary expression profiles of source genes of the present inventionthat are highly over-expressed or exclusively expressed in melanoma areshown in FIGS. 2A-2C. Expression scores for further exemplary genes areshown in Table 9.

TABLE 9 Expression scores. The table lists peptides from genes that arevery highly over-expressed in tumors compared to a panel of normaltissues (+++), highly over-expressed in tumors compared to a panel ofnormal tissues (++) or over-expressed in tumors compared to a panel ofnormal tissues (+). The baseline for this score was calculated frommeasurements of the follow relevant normal tissues: adipose tissue,adrenal gland, artery, blood cells, bone marrow, brain, cartilage,colon, esophagus, eye, gallbladder, head-and-neck and salivary gland,heart, kidney, liver, lung, lymph node, pancreas, peripheral nerve,pituitary, rectum, skeletal muscle, skin, small intestine, spleen,stomach, thyroid gland, trachea, urinary bladder, and vein. In caseexpression data for several samples of the same tissue type wereavailable, the arithmetic mean of all respective samples was used forthe calculation. SEQ ID No Sequence GeneExpression 2 VLLGENVEL +++ 3VLFKDPVSV + 4 KTWDQVPFSV +++ 5 ILDEGHILQL +++ 6 SIPDTIASV +++ 9SLAGLVLYV +++ 12 RLDDKTTNV ++ 13 IQSETTVTV +++ 14 VLYEMLYGL + 18SLADVVDTL + 20 VISPHGIASV +++ 25 FIDPEQIQV +++ 33 SLYSYFQKV +++ 35ALFAAGANV +++ 38 KLGFGTGVNVYL +++ 39 ALPPPPASI +++ 40 LLSNTVSTL +++ 41LLDDPTNAHFI +++ 42 VLKADVVLL +++ 43 LLPDPLYSL 44 FLYTYIAKV +++ 45FVYGEPREL +++ 46 VMSSTLYTV +++ 47 ALDSDPVGL ++ 48 HLIGWTAFL +++ 49ALLSQDFEL +++ 50 HLDQIFQNL ++ 52 NLDYAILKL ++ 55 NLDKLYHGL + 57GILSFLPVL +++ 58 ILGDWSIQV ++ 61 KLSVHVTAL + 71 SLDEVAVSL + 125YLFAFLNHL + 126 SLIDFVVTC ++ 171 KVSGVILSV ++

Example 3 In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for HLA-A*0201 restricted TUMAPs ofthe invention, demonstrating that these peptides are T-cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 10).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nürnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 339) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.340), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for Melanoma Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 2peptides of the invention are shown in FIGS. 3A and 3B together withcorresponding negative controls. Additional exemplary flow cytometryresults after TUMAP-specific multimer staining for 3 peptides of theinvention are shown in FIGS. 4A through 4C together with correspondingnegative controls. Results for 33 peptides from the invention aresummarized in Table 10A. Additional results for 29 peptides from theinvention are summarized in Table 10B.

TABLE 10A in vitro immunogenicity of HLA class I peptides of theinvention Exemplary results of in vitro immunogenicity experimentsconducted by the applicant for the peptides of the invention. <20% = +;20%-49% = ++; 50%-69% = +++; >=70% = ++++ Seq ID No Peptide CodeSequence Wells Donors 238 FMN1-001 KLLDKPEQFL + ++ 241 MYO10-002RLYTKLLNEA +++ ++++ 243 HSF2B-001 ALAGIVTNV + ++++ 247 NOL11-001ALLNAILHSA + ++++ 248 MAGED2-003 GLFAGLGGAGA + ++++ 250 AURKB-001RVLPPSALQSV + +++ 252 TOP2A-002 YLLDMPLWYL + ++++ 254 SHCB-001FLMKNSDLYGA + ++++ 257 NCAPG-005 VLLNEILEQV ++ ++++ 262 IL8-001KLAVALLAA ++ ++ 264 GYG2-001 KVFDEVIEV + + 267 PTCD2-001 LLTDNVVKL +++++ 269 CEP55-001 ALNESLVEC + ++++ 271 ECT2-001 SLVQRVETI + ++ 277KIF18A-001 KTASINQNV +++ ++++ 278 SIX4-001 SLITGQDLLSV + ++++ 283MMP1-003 YTFSGDVQL + ++++ 287 CHEK1-001 KISDFGLATV ++ ++++ 292 MYBPH-001ALGDKFLLRV + ++++ 294 SMC2-001 FLLAEDTKV ++ ++++ 298 CENPE-001KLQEEIPVL + ++++ 308 TMEM43-001 KLLSDPNYGV + ++++ 310 IFT81-001ALASVIKEL + ++ 315 CERC-001 KLSWDLIYL ++ ++++ 318 ATAD5-002 SIIEYLPTL +++++ 320 MSH6-001 KIIGIMEEV ++++ ++++ 321 ELOVL2-001 YLPTFFLTV ++ +++322 ATP-001 SLHFLILYV ++ +++ 323 C11orf24-001 VVDKTLLLV +++ ++++ 326MCM5-001 ALSGTLSGV + ++++ 328 ZNF318-001 SLSQELVGV + ++ 332 DROSHA-001AVVEFLTSV + ++ 336 MET-001 YVDPVITSI ++ ++++

TABLE 10B In vitro immunogenicity of HLA class I peptides of theinvention Exemplary results of in vitro immunogenicity experimentsconducted by the applicant for HLA-A*02 restricted peptides of theinvention. Results of in vitro immunogenicity experiments are indicated.Percentage of positive wells and donors (among evaluable) are summarizedas indicated <20% = +; 20%-49% = ++; 50%-69% = +++; >=70% = ++++ SEQ IDNo Sequence Wells positive [%] 3 VLFKDPVSV + 4 KTWDQVPFSV + 8 SIIPYLLEA++ 9 SLAGLVLYV ++ 10 KMTQYITEL ++ 11 TLIELLLPKL + 12 RLDDKTTNV ++ 13IQSETTVTV ++ 14 VLYEMLYGL +++ 15 VLYDPVVGC + 16 GLFPSNFVTA + 17GVVHGVATV + 18 SLADVVDTL + 20 VISPHGIASV ++ 21 FMYNFQLVTL + 31 FLPRKFPSL++ 33 SLYSYFQKV ++++ 34 YLINFEIRSL + 116 LMDETMKEL + 121 FLEDLVPYL +++128 ALFPGDVDRL + 133 ALDPAYTTL + 155 GLAGFFASV ++++ 189 KALQFLEEV + 191YLDKMNNNI + 192 KLFTQIFGV + 211 FLPFLTTEV + 213 LLDPTNVFI ++ 232KILPDLNTV +

Example 4 Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizes (trifluoro acetate salt) in purities of >50%. All TUMAPs arepreferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5 MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 11 MHC class I binding scores. Binding of HLA-class I restrictedpeptides to HLA-A*02:01 was ranged by peptide exchange yield: >10%= +; >20% = ++; >50 = +++; >75% = ++++ SEQ ID No Sequence Peptideexchange 1 FLDVKELML ++++ 2 VLLGENVEL +++ 3 VLFKDPVSV ++++ 4 KTWDQVPFSV++++ 5 ILDEGHILQL ++++ 6 SIPDTIASV +++ 7 NLQEKVPEL +++ 8 SIIPYLLEA ++++9 SLAGLVLYV ++++ 10 KMTQYITEL ++++ 11 TLIELLLPKL ++++ 12 RLDDKTTNV +++13 IQSETTVTV ++++ 14 VLYEMLYGL ++++ 15 VLYDPVVGC ++++ 16 GLFPSNFVTA ++++17 GVVHGVATV ++++ 18 SLADVVDTL ++++ 19 VLAVLGAVVAV +++ 20 VISPHGIASV++++ 21 FMYNFQLVTL ++ 22 KLLELQELVL ++++ 23 FLGDPPPGL +++ 24 SLVAILHLL+++ 25 FIDPEQIQV +++ 26 KIEDLIKYL +++ 27 TLWYVPLSL ++++ 28 IVDNTTMQL +++29 ILDDVAMVL +++ 30 VLFPMDLAL +++ 31 FLPRKFPSL ++++ 32 GLDIITNKV +++ 33SLYSYFQKV ++++ 34 YLINFEIRSL ++++ 35 ALFAAGANV +++ 36 SVNGFISTL ++ 37TLKEYLESL +++ 38 KLGFGTGVNVYL ++++ 39 ALPPPPASI +++ 40 LLSNTVSTL +++ 41LLDDPTNAHFI +++ 42 VLKADVVLL ++ 43 LLPDPLYSL ++ 44 FLYTYIAKV +++ 45FVYGEPREL +++ 46 VMSSTLYTV ++++ 47 ALDSDPVGL +++ 48 HLIGWTAFL ++++ 49ALLSQDFEL ++++ 50 HLDQIFQNL ++ 51 LIDKIIEYL ++ 52 NLDYAILKL + 53ILDEEKFNV +++ 54 LLDSGAFHL +++ 55 NLDKLYHGL + 56 ILDELVKSL +++ 57GILSFLPVL +++ 58 ILGDWSIQV ++++ 59 IIDDVMKEL ++ 60 ILPEAQDYFL ++++ 61KLSVHVTAL ++++ 62 LLDTTQKYL ++++ 63 SIDDSDPIV + 64 SLGPIMLTKI ++ 65TTLGGFAKV ++ 66 VMFEYGMRL ++++ 67 YVDSEGIVRM + 68 FLAEAARSL ++++ 69IIDDKPIGL +++ 70 LIDEAAQML +++ 71 SLDEVAVSL ++++ 72 TLLEVDAIVNA ++++ 73ELDKIYETL + 74 GTIPLIESL + 75 FMYAGQLTL ++++ 76 QIDSIHLLL +++ 77SIDDVVKKL ++ 78 ALKDLVNLI ++++ 79 AVDNILLKL +++ 80 FADELSHLL +++ 81FLDDGNQML +++ 82 GIDDLHISL +++ 83 GLDKVITVL +++ 84 GLDTILQNL ++++ 85GLLDVMYQV ++++ 86 HTLPHEIVVNL +++ 87 IIDPPLHGQLL ++ 88 ILDGIIREL +++ 89ILDNSPAFL +++ 90 ILDYIHNGL +++ 91 ILLDRLFSV ++++ 92 KLPGFPTQDDEV ++ 93LLAKAVQNV +++ 94 LLDAFSIKL +++ 95 LLDALQHEL +++ 96 LLDMSLVKL +++ 97NLDATVTAL +++ 98 NLPNTNSILGV +++ 99 NLPSELPQL +++ 100 NLREILQNV +++ 101NVDENVAEL ++ 102 RLPDQFSKL +++ 103 SLDAVMPHL +++ 104 SLDQIIQHL +++ 105SLKQTVVTL +++ 106 TLSEICEFI ++++ 107 TLVAFLQQV ++++ 108 TVIRPLPGL ++ 109VIDDLIQKL ++ 110 VLDTLTKVL +++ 111 VLDVSFNRL +++ 112 VLPAVLTRL +++ 113VLYSLVSKI +++ 114 VVDDIVSKL ++ 115 YIDDVFMGL +++ 116 LMDETMKEL ++++ 117KQQASQVLV +++ 118 TMIEICEKL ++++ 119 SLGLGFISRV +++ 120 QLMEGKVVL ++++121 FLEDLVPYL ++++ 122 YVDDFGVSV +++ 123 LLGEGIPSA ++++ 124 FLPQKIIYL++++ 125 YLFAFLNHL ++++ 126 SLIDFVVTC +++ 127 TLISDIEAVKA +++ 128ALFPGDVDRL +++ 129 VLPDDLSGV +++ 130 GLVDVLYTA +++ 131 FVDPNGKISL +++132 FLDASGAKL ++++ 133 ALDPAYTTL +++ 134 LLDEVLHTM ++++ 135 FLDDQETRL+++ 136 FAYDGKDYIAL +++ 137 ILPSNLLTV +++ 138 YLDKTFYNL +++ 139AVDATVNQV +++ 140 RLEAYLARV +++ 141 YVIDPIKGL +++ 142 FVDGSAIQV +++ 143ILDDSALYL ++++ 144 SVDEVEISV +++ 145 TLPNIYVTL +++ 146 GVGPVPARA +++ 147ILDDQTNKL +++ 148 TLKDIVQTV +++ 149 YLDTFALKL ++++ 150 KLFPSPLQTL ++++151 FLGEPASYLYL ++++ 152 IMEDFTTFL ++++ 153 RLDEVSREL +++ 154 TLGTATFTV++++ 155 GLAGFFASV ++++ 156 ALMDTDGSGKLNL +++ 157 HLFETISQA +++ 158KLIPSIIVL +++ 159 TILATVPLV ++++ 160 ALDDISESI ++++ 161 GLCDSIITI ++++162 TLDGNPFLV +++ 163 RLMANPEALKI +++ 164 ALFFQLVDV ++ 165 ALIEVLQPLI++++ 166 SIIPPLFTV ++++ 167 KVLGDVIEV ++++ 168 KLLAATLLL ++++ 169TLLESIQHV ++++ 170 KLKEAVEAI ++ 171 KVSGVILSV ++++ 172 FLPAGIVAV ++++173 ALDDIIYRA +++ 174 TLLEGLTEL +++ 175 VLDSVDVRL ++++ 176 TLYEQEIEV++++ 177 ILWDTLLRL ++++ 178 FAYDGKDYIA ++++ 179 ALDDTVLQV +++ 180KLAEALYIA +++ 181 GLIDLEANYL ++++ 182 SVALVIHNV ++++ 183 FLDSLIYGA ++++184 VLFSSPPVILL ++++ 185 ILADATAKM ++++ 186 FLDHEMVFL ++++ 187 SLPRPTPQA+++ 188 VVVDPIQSV +++ 189 KALQFLEEV ++++ 191 YLDKMNNNI ++++ 192KLFTQIFGV ++++ 193 ALDEPTTNL +++ 194 TLDDIMAAV +++ 195 IAAGIFNDL + 196ALEPIDITV +++ 197 ALDSGFNSV ++++ 198 EVVDKINQV + 199 AIHTAILTL ++ 200LLEEINHFL +++ 201 SLIDRTIKM +++ 202 RVAFKINSV +++ 203 FLNEDISKL +++ 204RMDEEFTKI +++ 205 SLKSKVLSV ++++ 206 LLYEDIPDKV +++ 207 VQIGDIVTV ++++208 YSDDIPHAL ++ 209 SILDGLIHL +++ 210 LLPELRDWGV +++ 211 FLPFLTTEV ++++212 LLKDSIVQL +++ 213 LLDPTNVFI ++++ 214 VLMEMSYRL +++ 215 EVISKLYAV +++216 TLLHFLAEL ++++ 217 NMMSGISSV +++ 218 STLHLVLRL +++ 219 FLDSEVSEL +++220 SAAEPTPAV +++ 221 SLLPTEQPRL +++ 222 LLSEIEEHL ++++ 223 FLETNVPLL+++ 224 ILDEPTNHL ++ 225 VLFGAVITGA ++++ 226 VLNEYFHNV ++++ 227FLLEQEKTQAL ++++ 228 FLNLFNHTL ++++ 229 LLEPFVHQV ++++ 230 HLDEARTLL++++ 231 KMVGDVTGA +++ 232 KILPDLNTV ++++ 233 QLYNQIIKL ++++ 234KVPEIEVTV ++++ 235 ALADLQEAV ++++ 236 GLDSGFHSV ++++ 237 VLYNESLQL ++++

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1. A method for treating a patient who has cancer comprisingadministering to the patient a population of activated T cells thatselectively recognize cancer cells that present a peptide consisting ofthe amino acid sequence of ILLDRLFSV (SEQ ID NO: 91), wherein saidcancer is selected from the group consisting of head-and-neck cancer,melanoma, acute myelogenous leukemia, breast cancer, bile duct cancer,brain cancer, chronic lymphocytic leukemia, colorectal carcinoma,esophageal cancer, gallbladder cancer, gastric cancer, hepatocellularcancer, non-Hodgkin lymphoma, non-small cell lung cancer, ovariancancer, pancreatic cancer, prostate cancer, renal cell cancer, smallcell lung cancer, urinary bladder cancer, and uterine cancer.
 2. Themethod of claim 1, wherein the T cells are autologous to the patient. 3.The method of claim 1, wherein the T cells are obtained from a healthydonor.
 4. The method of claim 1, wherein the activated T cells areproduced by contacting T cells with the peptide loaded human class I orII MHC molecules expressed on the surface of an antigen-presenting cellfor a period of time sufficient to activate the T cells.
 5. The methodof claim 1, wherein the activated T cells are expanded in vitro.
 6. Themethod of claim 1, wherein the peptide is in a complex with an MHC classI molecule.
 7. The method of claim 4, wherein the antigen presentingcell is infected with a recombinant virus expressing the peptide.
 8. Themethod of claim 7, wherein the antigen presenting cell is a dendriticcell or a macrophage.
 9. The method of claim 5, wherein the expansion isin the presence of an anti-CD28 antibody and IL-12.
 10. The method ofclaim 1, wherein the population of activated T cells comprisesCD8-positive cells.
 11. The method of claim 4, wherein the contacting isin vitro.
 12. The method of claim 1, wherein the population of activatedT cells are administered in the form of a composition.
 13. The method ofclaim 12, wherein the composition comprises an adjuvant.
 14. The methodof claim 13, wherein the adjuvant is selected from the group consistingof anti-CD40 antibody, imiquimod, resiguimod, GM-CSF, cyclophosphamide,Sunitinib, bevacizumab, interferon-alpha, interferon-beta, CpGoligonucleotides and derivatives, poly-(I:C) and derivatives, RNA,sildenafil, and particulate formations with poly(lactid co-glycolid)(PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13,IL-15, IL-21, and IL-23.
 15. The method of claim 1, wherein the canceris head-and-neck cancer.
 16. A method of eliciting an immune response ina patient who has cancer comprising administering to the patient apopulation of activated T cells that selectively recognize cancer cellsthat present a peptide consisting of the amino acid sequence ofILLDRLFSV (SEQ ID NO: 91), wherein said cancer is selected from thegroup consisting of head-and-neck cancer, melanoma, acute myelogenousleukemia, breast cancer, bile duct cancer, brain cancer, chroniclymphocytic leukemia, colorectal carcinoma, esophageal cancer,gallbladder cancer, gastric cancer, hepatocellular cancer, non-Hodgkinlymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer,prostate cancer, renal cell cancer, small cell lung cancer, urinarybladder cancer, and uterine cancer.
 17. The method of claim 16, whereinthe activated T cells are produced by contacting T cells with thepeptide loaded human class I or II MHC molecules expressed on thesurface of an antigen-presenting cell for a period of time sufficient toactivate the T cells.
 18. The method of claim 16, wherein the immuneresponse comprises a cytotoxic T cell response.
 19. The method of claim16, wherein the immune response is capable of killing cancer cells thatpresent a peptide consisting of the amino acid sequence of ILLDRLFSV(SEQ ID NO: 91).
 20. The method of claim 16, wherein the cancer ishead-and-neck cancer.